Tissue engineering methods and compositions

ABSTRACT

The presently disclosed subject matter generally relates to methods and systems for facilitating the growth and differentiation of adipose-derived stem cells for laboratory and therapeutic applications. The cells can be employed alone or in conjunction with unique biologically-compatible scaffold structures to generate differentiated tissues and structures, both in vitro and in vivo. The presently disclosed subject matter further relates to methods of forming and using improved tissue engineered scaffolds that can be used as substrates to facilitate the growth and differentiation of cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. Nos. 60/715,530 and 60/724,044, filed Sep. 9, 2005 andOct. 6, 2005, respectively, the disclosures of which are herebyincorporated by reference in their entireties.

GRANT STATEMENT

This work was supported by grants AR49294 and GM08555 from the UnitedStates National Institutes of Health. Thus, the U.S. government hascertain rights in the presently disclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter generally relates to novelmethods and systems for differentiating adipose derived stem (ADS) cellsto provide cells and tissues suitable for laboratory and therapeuticapplications. These cells, and other cells, can be employed alone or inconjunction with unique biologically-compatible scaffold structures togenerate differentiated tissues and structures, both in vitro and invivo.

TABLE OF ABBREVIATIONS

-   -   ADS—adipose-derived stem    -   ANOVA—analysis of variance    -   BaCl₂—barium chloride    -   BMI—body mass index    -   BMP—bone morphogenic protein    -   CaCl₂—calcium chloride    -   CAT—chloramphenicol acetyl transferase    -   cm—centimeter    -   CMV—cytomegalovirus    -   COMP—cartilage oligomeric protein    -   Dhfr—dihydrofolate reductase    -   DMEM—Dulbecco's Modified Eagle's Medium    -   DNA—deoxyribonucleic acid    -   dsDNA—double stranded DNA    -   EG—embryonic germ    -   EGF—epidermal growth factor    -   ePTFR—expanded PTFE    -   ES—embryonic stem    -   FACS—fluorescence-activated cell sorting    -   FBS—fetal bovine serum    -   FGF—fibroblast growth factor    -   g—gram    -   GAG—glycosaminoglycan    -   GF—growth factor    -   GFP—green fluorescent protein    -   GLA—glycine leucine alanine    -   hADS—human adipose-derived stem    -   Hprt—hypoxanthine phosphoribosyl transferase    -   HSC—hematopoietic stem cells    -   HSV-tk—herpes simplex virus-thymidine kinase    -   IEp—immediate early viral promoter    -   IGF-I—insulin-like growth factor    -   ITR—inverted terminal repeat    -   L—liter    -   LTR—long terminal repeat    -   mg—milligram    -   mL—milliliter    -   MLV—murine leukemia virus    -   mM—millimolar    -   mRNA—messenger RNA    -   MSC—mesenchymal stem cell    -   NaCl—sodium chloride    -   ng—nanogram    -   p—probability    -   PBS—phosphate buffered saline    -   PCR—polymerase chain reaction    -   PEEK—polyetheretherketone    -   PEG—polyethylene glycol    -   PEO—polyethylene oxide    -   PGA—polyglycolic acid    -   PDGF—platelet-derived growth factor    -   Pgk—phosphoglycerate kinase    -   PLA—processed lipoaspirate    -   PLSD—protected least significant difference    -   PTFE—polyetetrafluoroethylene    -   PTHrP—parathyroid hormone-related protein    -   RGD—arginine glycine aspartic acid    -   RNA—ribonucleic acid    -   RSV—rous sarcoma virus    -   S.E.M.—standard error of measurement    -   SV40—simian virus 40    -   TAF—transcription associated factor    -   TGF-α—transforming growth factor α    -   TGF-β—transforming growth factor β    -   VEGF—vascular endothelial growth factor    -   2-D—2-dimensional    -   3-D—3-dimensional    -   ³H—tritium    -   μCi—microcuries    -   μg—microgram    -   μm—micrometer    -   %—percent    -   #—number    -   ≦—less than or equal to    -   ≧—greater than or equal to    -   >—greater than    -   <—less than    -   =—equal to    -   ±—plus or minus    -   +—plus

BACKGROUND

Many disease conditions or injuries of the body require the repair orreplacement of damaged tissues and/or structures, but the body itselfmay not be able to replace or repair the tissue and/or structuressatisfactorily or within an appropriate time scale. Accordingly, manymethods of disease or injury treatment involve augmenting the body'snatural repair mechanisms and often rely on the use of implantablebiological scaffolds or prostheses. Tissue engineering attempts tocreate three-dimensional tissue structures on which cells and otherbiomolecules can be incorporated. These structures or scaffolds guidethe organization, growth and differentiation of cells in the process offorming functional tissue by providing physico-chemical cues.

For example, degenerative joint diseases such as osteoarthritis remain asource of significant pain and disability, resulting in an economicburden of over 40 billion dollars per year to the United States. Presenttreatment options for osteoarthritis are limited, and surgicalmanagement generally involves replacement of the joint with a metal andpolyethylene prosthesis. The short life span and loading tolerance ofjoint replacement makes this treatment unacceptable for young,potentially active individuals. The treatment of synovial joints usingtissue engineered grafts shows tremendous promise but its applicationhas been limited to the treatment of small cartilage defects in the kneejoint.

Further, articular cartilage is avascular, aneural, and has limitedcapacity for self-repair. Particularly, articular cartilage is a thinlayer of soft connective tissue (0.5-5 mm thick) that covers thearticulating surfaces of long bones in synovial joints. The principalfunction of articular cartilage is to redistribute applied loads and toprovide a flow friction-bearing surface to facilitate movement withinthese joints. Damage to this connective tissue in joints results insignificant pain and morbidity, and currently, there are few optionsavailable for treatment. Some treatment options include lavage,debridement, microfracture, and autologous and/or allogeneicosteochondral/chondral grafts (reviewed in Hunziker (2002)Osteoarthritis Cartilage 10:432-463.

The success rates from these treatment options vary greatly, and someshow promise. However, in many of the studies, the results suggestfibrous tissue formation, apoptosis, and further cartilage degenerationnonetheless occur (Furukawa et al. (1980) J Bone Joint Surg Am 62:79-89;Kim et al. (1991) J Bone Joint Surg Am 73:1301-1315; Shapiro et al.(1993) J Bone Joint Surg Am 75:532-553; Nehrer et al. (1999) Clin OrthopRelat Res 365:149-162; Tew et al. (2000) Arthritis Rheum 43:215-225;Mitchell and Shephard (2004) Clin Orthop Relat Res 423:3-6. Autologouschondrocyte transplants studies have also shown an inability to producehyaline cartilage repair tissue, specifically over long time periodsBrittberg et al. (1996) Clin Orthop Relat Res 326:270-283; Brittberg(1999) Clin Orthop Relat Res 367(Suppl):S147-155; Nehrer et al. (1999)Clin Orthop Relat Res 365:149-162; Breinan et al. (2001) J Orthop RelatRes 19:482-492, and even though some clinical studies have shown somepromising results Brittberg et al. (1994) N Engl J Med 331:889-895;Breinan et al. (1997) J Bone Joint Surg Am 79:1439-1451; Minas andNehrer (1997) Orthopedics 20:525-538; Gillogly et al. (1998) J OrthopSports Phys Ther 28:241-251, as with the other treatment options,randomized, controlled trials are needed to truly ascertain the efficacyof these procedures. Given the success rate to date of current cartilageremodeling, repair, regrowth, and/or regeneration treatment options,combined with the burgeoning economic burden cartilage pathology andosteoarthritis has on society (Jackson et al. (2001) Clin Orthop RelatRes 391(Suppl):S14-25), novel tissue engineering approaches are neededto establish improved options for the treatment of cartilage defects andosteoarthritis, among other maladies.

In recent years, the identification of mesenchymal stem cells has led toadvances in tissue regrowth and differentiation. Such cells arepluripotent cells found in bone marrow and periosteum, capable ofdifferentiating into various mesenchymal or connective tissues. Forexample, such bone-marrow derived stem cells can be induced to developinto myocytes upon exposure to agents such as 5-azacytidine (Wakitani etal. (1995) Muscle Nerve, 18(12), 1417-26). It has been suggested thatsuch cells are useful for repair of tissues such as cartilage, fat, andbone (see, e.g., U.S. Pat. Nos. 5,908,784, 5,906,934, 5,827,740,5,827,735), and that they also have applications through geneticmodification (see, e.g., U.S. Pat. No. 5,591,625). While theidentification of such cells has led to advances in tissue regrowth anddifferentiation, the use of such cells is hampered by several technicalhurdles. One drawback to the use of such cells is that they are veryrare (representing as few as 1/2,000,000 cells), making any process forobtaining and isolating them difficult and costly. Additionally, bonemarrow harvest is universally painful to the donor. Moreover, such cellsare difficult to culture without inducing differentiation, unlessspecifically screened sera lots are used, adding further cost and laborto the use of such stem cells. Thus, there is a need for a more readilyavailable source for pluripotent stem cells, particularly cells that canbe cultured without the requirement for costly prescreening of culturematerials.

Other advances in tissue engineering have shown that cells can be grownin specially-defined cultures to produce three-dimensional structures.Spatial definition typically is achieved by using various acellularfiber scaffolds or matrices to support and guide cell growth anddifferentiation. While this technique is still in its infancy,experiments in animal models have demonstrated that it is possible toemploy various acellular fiber scaffold materials to regenerate wholetissues (see, e.g., Probst et al. (2000) BJU Int., 85(3), 362-367).While artificial fiber scaffolds have been developed, these can provetoxic either to cells or to patients when used in vivo, or do notprovide adequate mechanical support required for tissue repair.Accordingly, there remains a need for a scaffold material suitable foruse as a substrate in culturing and growing populations of cells,wherein the matrix, cell combination is tailored specifically forreplacement of a target tissue. Ultimately, this replacement tissue willserve to substantially function as the native tissue it seeks toreplace.

Accordingly, the presently disclosed subject matter addresses needs inthe art for improved methods for producing improved tissue engineeredimplantable compositions. This and other needs are addressed in whole orin part by the presently disclosed subject matter.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

Methods and compositions for treating a tissue pathology in a subjectare disclosed. In some embodiments, the subject is a mammalian subject.

In some embodiments, the method comprises providing to anadipose-derived stem (ADS) cell in culture; exposing the ADS cell to aneffective amount of a BMP-6 polypeptide or a functional fragmentthereof, wherein the effective amount of the BMP-6 polypeptide or thefunctional fragment thereof is sufficient to induce the ADS cell todifferentiate into a cell capable of treating the tissue pathology inthe subject; administering the cell to the subject. In some embodiments,the effective amount of BMP-6 ranges from about 1 picogram/mL to about10 milligram/mL.

Also disclosed is a method for treating a tissue pathology in a subject,comprising providing to an adipose-derived stem (ADS) cell in culture;exposing the ADS cell to an effective amount of a biologically activeagent, wherein the effective amount of the biologically active agent issufficient to induce the ADS cell to differentiate into a cell capableof treating the tissue pathology in the subject; and administering thecell to the subject.

Also disclosed is a composition for treating a tissue pathology in asubject. The composition can comprise an adipose-derived stem (ADS) cellthat has been differentiated in vitro by exposure to an effective amountof a BMP-6 polypeptide or a functional fragment thereof; and apharmaceutically acceptable carrier or excipient.

Also disclosed is composition for treating a tissue pathology in asubject, the composition comprising an adipose-derived stem (ADS) cell;an effective amount of a BMP-6 polypeptide or a functional fragmentthereof; and a pharmaceutically acceptable carrier or excipient.

Also disclosed is a composition for treating a tissue pathology in asubject, the composition comprising an adipose-derived stem (ADS) cellthat has been differentiated in vitro by exposure to an effective amountof a biologically active agent; and a pharmaceutically acceptablecarrier or excipient.

Also disclosed is a composition for treating a tissue pathology in asubject, the composition comprising an adipose-derived stem (ADS) cell;an effective amount of a biologically active agent; and apharmaceutically acceptable carrier or excipient.

The cell can be administered to a target tissue selected from the groupincluding but not limited to articular cartilage, non-articularcartilage, auricular cartilage, tracheal cartilage, laryngeal cartilage,nasal cartilage, growth plate cartilage, meniscus, labrum,intervertebral disc, tendon, ligament, periodontal ligament, fascia, andmuscle. The target tissue can comprise multiple tissue types that areintegrated with one another selected from the group consisting of boneand cartilage, muscle and tendon, and ligament and bone. The tissuepathology can comprise a compromise in the normal homeostasis of thetissue, optionally culminating in degeneration of the tissue. The tissuepathology can comprise loss, damage, injury, or combinations thereof tothe tissue. The treating can comprise tissue remodeling, repair,regrowth, resurfacing, regeneration, or combinations thereof.

The ADS cells from an adipose depot selected from the group can beselected from the group including but not limited to subcutaneousabdomen, thigh, buttocks, infrapatellar fat pad, and combinationsthereof. The ADS cell can be selected from the group including but notlimited ADS cells autologous to the subject, ADS cells allogeneic to thesubject, ADS cells xenogenic to the subject, and combinations thereof.

The isolated ADS cells can be transfected with an expression constructencoding a biologically active agent, such as but not limited to atleast one of BMP-6 polypeptide, a BMP-6 receptor polypeptide, or afunctional fragment thereof. The expression construct can comprise aregulatable promoter operatively linked to at least one coding sequence.The expression vector encoding a biologically active agent (such as butnot limited to a BMP-6 polypeptide or a functional fragment thereof) canbe administered in addition to the differentiated cell. The expressionvector can be selected from the group including but not limited to aviral vector, an adenovirus vector, an adeno-associated virus vector, aplasmid, and a deoxyribonucleic acid molecule.

The ADS cell can be present in or on a biocompatible scaffold. Thebiologically active agent, such as but not limited to a BMP-6polypeptide or functional fragment thereof, can be incorporated into thescaffold for controlled release over time.

The ADS cell can be exposed in culture to at least one otherbiologically active agent, such as but not limited to a growth factor orcytokine. Representative growth factors or cytokines including but arenot limited a TGF-β superfamily member, an IGF-1, an FGF, an EGF, aPDGF, a parathyroid hormone related peptide (PTHrP), an interleukin, andcombinations thereof.

Another cell type other than the ADS cell can be administered along withthe ADS cell. The other cell type can be selected from the groupincluding but not limited to a chondrocyte, a fibroblast, an osteoblast,a myoblast, a neuron, a progenitor cell, and combinations thereof.

A subpopulation of differentiated ADS cells can be selected. Thesubpopulation of differentiated ADS cells can be selected based on: (i)expression of at least one cell surface marker can be selected from thegroup including but not limited to CD10, CD13, CD31, CD34, CD36, CD44,CD49, CD54, CD55, CD59, CD65 CD105, and CD166; (ii) differentialexpression of aldehyde dehydrogenase (ALDH); (iii) differentialexpression of collagen 1; (iv) efflux of a dye such or a nucleic acidlabel; (v) telomere length or the expression of telomerase; (vi)expression of TGF-β superfamily members; (vii) expression of TGF-βsuperfamily member receptor polypeptides; or (viii) combinations of anyof the foregoing. The dye can comprise Hoechst 33342. The subpopulationof differentiated ADS cells can be selected by repeated passage inculture.

The differentiated ADS cell can be identified as a cell suitable for usein therapeutic restorative and regenerative techniques when geneexpression measurements, protein measurements, or combinations thereofmeet predetermined parameters.

The ADS cell can be passaged at least twice in culture, wherein thepassaging enhances an ability of the cell to express at least onemacromolecule associated with a predetermined connective tissue uponexposure to a biologically active agent, such as but not limited toBMP-6 or a functional fragment thereof.

Also disclosed is a joint resurfacing implant adapted for use with apredetermined joint. In some embodiments, the implant comprises abiocompatible scaffold, wherein the scaffold can resurface at least aportion of an articulating surface of the predetermined joint uponimplantation. In some embodiments, the implant comprises: a scaffoldcomprising a biocompatible material; and one or more cells, wherein thescaffold and one or more cells can resurface at least a portion of anarticulating surface of a predetermined joint upon implantation. In someembodiments, the implant comprises a cell-seeded biocompatible scaffold,wherein at least a fraction of the cells or scaffold is devitalizedbefore implantation, and wherein the scaffold can resurface at least aportion of an articulating surface of the predetermined joint uponimplantation. Methods for making and using the implants in jointresurfacing are also disclosed.

The biocompatible material can comprise a material selected from thegroup including but not limited to an absorbable material, anon-absorbable material, and combinations thereof. The non-absorbablematerial can be selected from the group including but not limited to apolytetrafluoroethylene (PTFE), an expanded PTFE (ePTFE), a polyamide, anylon, a polysulfone, a cellulosic, an acrylic, tantalum, polyvinylalcohol, carbon, ceramic, a metal, an acrylic, a polycarbonate, apolyester, a polyether, a poly(ether ketone), a poly(ether etherketone), a poly(aryl ether ketone), a poly(ether ether ketone etherketone), a poly(ethylene terephthalate), a poly(methyl (meth)acrylate),a polyolefin, a polysulfone, a polyurethane, a polyethylene, apolypropylene, a poly(vinyl chloride), a carbon fiber reinforcedcomposite, a glass fiber reinforced composite, and combinations thereof.The absorbable material can be selected from the group including but notlimited to a polyglycolic acid (PGA), a polylactic acid (PLA), apolyglycolide-lactide, a polycaprolactone, a polydioxanone, apolyoxalate, a polyanhydride, a poly(phosphoester), catgut suture,collagen, silk, alginate, agarose, chitin, chitosan, hydroxyapatite,bioabsorbable calcium phosphate, hyaluronic acid, elastin, apolyorthoester, a poly(amino acid), a pluronic/F-12, a poly(ethyleneoxide)/poly(ethylene glycol) (PEO/PEG), collagen, gelatin, a bloodderivative, plasma, synovial fluid, serum, fibrin, hyaluronic acid, aproteoglycan, elastin, and combinations thereof.

The scaffold can comprise biocompatible fibers. The fibers can comprisea monofilament fiber, a multifilament fiber, a hollow fiber, a fiberhaving a variable cross-section along its length, or a combinationthereof. A two-dimensional fiber scaffold can be utilized, comprisingany woven, non-woven, knitted, or braided fiber system. Athree-dimensional fiber scaffold can be utilized, comprising threeorthogonally woven fiber systems, a plurality of braided fiber systems,a plurality of circular woven fiber systems, or combinations thereof.

The scaffold can comprise a three-dimensional fiber scaffold, thescaffold comprising at least three systems of fibers, wherein (i) two ofthe three fiber systems define an upper layer, a lower layer, and amedial layer between the upper layer and the lower layer within thethree-dimensional fiber scaffold; (ii) one of the at least three fibersystems interconnects the upper layer, the lower layer and the mediallayer; and (iii) the at least three fiber systems each comprise abiocompatible material. The at least three fiber systems in at least oneof the upper, medial, and lower layers can define a plurality ofinterstices within the fiber scaffold. The interstices can comprise apore size ranging from about 1 μm to about 1,000 μm , optionally about10 μm to about 500 μm, optionally from about 25 μm to about 250 μm, oroptionally, from about 50 μm to about 125 μm.

The implant can comprise a shape that corresponds to a majority of anarticulating surface of the predetermined joint. The shape can besubstantially that of the native predetermined joint.

One or more surfaces of the scaffold can be coated with a biomateriallayer. The biomaterial layer can comprise a gel.

In some embodiments of the scaffold, the one or more cells can beselected from the group including but not limited to primary cells,undifferentiated progenitor cells, stem cells, and combinations thereof.The undifferentiated progenitor cells or stem cells can be selected fromthe group including but not limited to stem or progenitor cells derivedfrom adipose tissue, bone marrow, synovium, muscle, bone, cord blood,embryos, amniotic fluid, periosteum, and combinations thereof. Theprimary cells can include but are not limited to chondrocytes,osteoblasts, fibroblasts, fibrochondrocytes, and combinations thereof.

The implant can comprise a biologically active material. Thebiologically active material can be selected from the group includingbut not limited to a growth factor, a cytokine, a chemokine, a collagen,gelatin, laminin, fibronectin, thrombin, lipids, cartilage oligomericprotein (COMP), thrombospondin, fibrin, fibrinogen, Matrix-GLA(glycine-leucine-alanine) protein, chondrocalcin, tenascin, a mineral,an RGD (Arginine-Glycine-Aspartic Acid) peptide or RGD-peptidecontaining molecule, elastin, hyaluronic acid, a glycosaminoglycan, aproteoglycan, water, an electrolyte solution, and combinations thereof.

The predetermined joint can include but is not limited to a hip joint, aknee joint, a shoulder joint, an ankle joint, thumb joint, finger joint,wrist joint, neck joint, spine joint, toe joint, temporomandibularjoint, patella, and an elbow joint.

The joint resurfacing implant can be maintained in a bioreactor prior toimplantation for a time sufficient to provide tissue that can resurfaceat least a portion of an articulating surface of the predeterminedjoint.

In administering the implant, part or all tissues present at site of thejoint can be removed. The tissue to be removed can include but is notlimited to cartilage, bone, ligaments, meniscus, synovium, andcombinations thereof. An entire articulating surface of the joint can beresurfaced. At least a portion of one or more, two or more, etc.,articulating surfaces of the joint can be resurfaced in part or in all.At least a portion of all articulating surfaces of the joint can beresurfaced. All articulating surfaces of the joint can be resurfaced.

Accordingly, it is an object of the presently disclosed subject matterto provide novel tissue engineering methods and compositions. This andother objects are achieved in whole or in part by the presentlydisclosed subject matter.

An object of the presently disclosed subject matter having been statedabove, other objects and advantages of the presently disclosed subjectmatter will become apparent to those of ordinary skill in the art aftera study of the following Description, Drawings, and non-limitingExamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a bar graph of analyses of DNA content after 7 days inculture (n≧4/condition/donor). Data are expressed as mean percent of day0 DNA±S.E.M. Patterned bars represent donor 1, white bars representdonor 2, and black bars represent donor 3.

FIGS. 2A and 2B present bar graphs of biosynthetic activity of hADS from3 donors under various growth factor treatments. [³H]-proline DPM/μg DNA(FIG. 3A) and [³⁵S]-sulfate DPM/μg DNA (FIG. 3B) incorporation intoprotein were determined. The x-axis values for each Figure are the sameas in FIG. 2. All comparisons between growth factor conditions aresignificant at p<0.05. “+” indicates non-significance at p≧0.05(n≧4/condition/donor). Data are presented as mean±S.E.M.). Patternedbars represent donor 1, white bars represent donor 2, and black barsrepresent donor 3.

FIGS. 3-6 depict gene expression analyses for AGC1 (FIG. 4;≧4/condition/donor. *, p<0.0001 relative to Day 0 Control. #, p<0.0001relative to Base Medium at Day 7); COL1A1 (FIG. 5; ≧4/condition/donor.*, p<0.05 relative to Day 0 Control. #, p<0.0001 relative to Base Mediumat Day 7); COL2A1 (FIG. 6; ≧4/condition/donor. *, p<0.05 relative to Day0 Control. #, p<0.05 relative to Base Medium at Day 7); and COL10A1(FIG. 7; ≧4/condition/donor. *, p<0.005 relative to Day 0 Control. #,p<0.05 relative to Base Medium at Day 7). For each of FIGS. 4-7, dataare presented as mean±S.E.M. For each of FIGS. 4-7, “o”, “□”, and “Δ”represent Donors 1, 2, and 3, respectively.

FIGS. 7A-7L depict photographs of the results of immunohistochemistry ofhADS cell-alginate beads after 7 days in culture. All photographs wereat 63× magnification. FIGS. 7A-7C demonstrate that three conditions(TGF-β1+Dex, TGF-β3+IGF-1+BMP-6, and BMP-6 only, respectively) showedincreased type I collagen staining over other conditions. FIGS. 7D-7Fdemonstrate that type X collagen expression decreases with BMP-6 withBMP-6 only having the least expression. FIGS. 7G-7I demonstrate that allconditions showed increased type II collagen staining over control, butthose with BMP-6 also have strongly staining matrix. FIGS. 7J-7Ldemonstrate that only those conditions with BMP-6 showed significantstaining of chondroitin sulfate with 3B3 antibody.

FIG. 8 is a diagram of the steps of one embodiment of the presentlydisclosed subject matter, involving the formation of a bioartificial hipimplant from autologous stem cells.

DETAILED DESCRIPTION

The presently disclosed subject matter provides methods and compositionsfor treating tissue pathologies in a subject, and methods for making thecompositions. In some embodiments an implantable composition comprisingone or more cells that can develop into one or more tissues at apredetermined site for treatment in the subject is provided. Treatmentcan be accomplished by implanting the composition at the predeterminedsite.

In some embodiments, the predetermined tissue types include but are notlimited to bone and cartilage; muscle and tendon, and ligament and bone.In some embodiments the predetermined site comprises the resurfacing ofthe articulating surface in a joint. In any of the presently disclosedembodiments, the sites for the intended replacement tissue can replacemultiple tissue types with one implantation (e.g. one tissue replacementto replace bone, cartilage, and the interface of bone and cartilage).

In some embodiments the tissue pathology can comprise a compromise inthe normal homeostasis of the tissue, optionally culminating indegeneration of the tissue. The tissue pathology can comprise loss,damage, degeneration, injury, or combinations thereof to the tissue. Thetreatment can comprise tissue remodeling, repair, regrowth, replacement,regeneration, or combinations thereof.

In some embodiments, the predetermined site comprises a target tissueselected from the group including but not to limited articularcartilage, non-articular cartilage, auricular cartilage, trachealcartilage, laryngeal cartilage, nasal cartilage, growth plate cartilage,meniscus, labrum, and intervertebral disc. Representative tissue typesat the predetermined site also include but are not limited tomusculoskeletal or dental connective tissues selected from the groupincluding but not limited to tendon, ligament, periodontal ligament,fascia, and muscle.

In some embodiments, the treatment is solely focused on the treatment ofthe articular surface of a joint.

To produce the desired implants, the compositions of the presentlydisclosed subject matter can be maintained under conditions suitable forthem to expand and divide to form the desired structures. In someapplications, this is accomplished by transferring the compositions to asubject (i.e., in vivo) typically at a site at which the new matter isdesired. Thus, for example, the presently disclosed subject matter canfacilitate the regeneration of tissues within an animal where thecompositions are implanted into such tissues. In other embodiments, thecompositions can be prepared in vitro. For examples cells present in thecompositions can be induced to differentiate and expand into tissues invitro. In such applications, the cells can be cultured on substrates orscaffolds that facilitate formation into three-dimensional structuresconducive for tissue formation.

I. Definitions

All technical and scientific terms used herein, unless otherwise definedbelow, are intended to have the same meaning as commonly understood byone of ordinary skill in the art. References to techniques employedherein are intended to refer to the techniques as commonly understood inthe art, including variations on those techniques or substitutions ofequivalent techniques that would be apparent to one of skill in the art.While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Following long-standing patent law tradition, the terms “a”, “an”, and“the” are meant to refer to one or more as used herein, including theclaims. For example, the phrase “a cell” can refer to one or more cells.

The term “absorbable” is meant to refer to a material that tends to beabsorbed by a biological system into which it is implanted.Representative absorbable fiber materials include, but are not limitedto polyglycolic acid (PGA), polylactic acid (PLA),polyglycolide-lactide, polycaprolactone, polydioxanone, polyoxalate, apolyanhydride, a poly(phosphoester), catgut suture, collagen, silk,chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate,hyaluronic acid, and any other medically acceptable yet absorbablefiber. Other absorbable materials include collagen, gelatin, a bloodderivative, plasma, synovial fluid, serum, fibrin, hyaluronic acid, aproteoglycan, elastin, and combinations thereof.

The term “non-absorbable” is meant to refer to a material that tends notto be absorbed by a biological system into which it is implanted.Representative non-absorbable fiber materials include but are notlimited to polypropylene, polyester, polytetrafluoroethylene (PTFE) suchas that sold under the registered trademark TEFLON® (E.I. DuPont deNemours & Co., Wilmington, Del., United States of America), expandedPTFE (ePTFE), polyethylene, polyurethane, polyamide, nylon,polyetheretherketone (PEEK), polysulfone, a cellulosic, fiberglass, anacrylic, tantalum, polyvinyl alcohol, carbon, ceramic, a metal (e.g.,titanium, stainless steel), and any other medically acceptable yetnon-absorbable fiber.

As used herein, the phrases “adipose-derived stem cell” and “ADS cell”refer to a cell with, at a minimum, unipotent potential that can beisolated from adipose tissue and that can be differentiated alongvarious mesodermal and ectodermal lineages. Representative conditionsare disclosed herein and have been described in the art, such as in U.S.Pat. No. 6,777,231 or Zuk et al. (2002) Mol Biol Cell 13:4279-4295, theentire contents of each of which are incorporated herein by reference.Adipose-derived stem cells can be isolated using techniques described inthese references. In some embodiments, an ADS cell can be isolated froma subject by removing subcutaneous fat from the subject, for example byliposuction. In some embodiments, an adipose-derived stem cell isisolated from a human, in which case it is referred to herein as a humanadipose-derived stem (hADS) cell.

As used herein, the terms “anisotropic”, “anisotropy”, and grammaticalvariations thereof, refer to properties of a scaffold and/or fibersystem as disclosed herein that can vary along a particular direction.Thus, the fiber and/or scaffold can be stronger and/or stiffer in onedirection versus another. In some embodiments, this can be accomplishedby changing fibers (such as, but not limited to providing fibers ofdifferent materials) in warp versus weft directions, and/or in the Zdirection, for example. Thus, anisotropic characteristics parallelnative properties of a tissue, and it is desirable to match orapproximate one or more native properties of the tissue in theimplantable composition.

Thus, strength can be provided in the direction needed and indeed it ispossible to restore properties of a tissue almost immediately withoutnecessarily needing for cells to grow into functional tissues. However,in some embodiments cells are provided and the growth into functionaltissues is also provided. Further, in some embodiments the scaffold cancomprise materials at least some, if not all of which, are absorbablematerials, such that degradation of the scaffold occurs over time. Thus,in some embodiments, the scaffold is replaced by tissue over time in thesubject.

In some embodiments, the terms “anisotropic”, “anisotropy” andgrammatical variations thereof, can also include, but is not limited tothe provision of more fiber in a predetermined direction. This can thusinclude a change of diameter in a fiber over a length of the fiber, achange in diameter at each end of the fiber, and/or a change in diameterat any point or section of the fiber; a change in cross-sectional shapeof the fiber; a change in density or number of fibers in a volumetricsection of the scaffold; and the use of monofilament fibers and/ormultifilament fibers in a volumetric section of the scaffold; and caneven include the variation in material from fiber system to fiber systemand along individual fibers in a volumetric section of the scaffold.

As used herein, the term “bioartificial” can refer to an implantablecomposition that comprises cells that were isolated, grown, and/ormanipulated in vitro, or the progeny of such cells. In some embodiments,a bioartificial joint replacement implant as disclosed herein comprisesa three-dimensional fiber scaffold and one or more cells that candevelop into tissues functioning substantially as bone, cartilage, bothbone and cartilage, or other tissues. In some embodiments, abioartificial joint replacement implant as disclosed herein comprises ascaffold which is partly or wholly acellular. In some embodiments, abioartificial joint replacement implant as disclosed herein comprises ascaffold that has been partly or wholly decellularized or devitalized atsome point in time after being seeded with cells.

The terms “biocompatible” and “medically acceptable” are usedsynonymously herein and are meant to refer to a material that iscompatible with a biological system, such as that of a subject having atissue (e.g., a joint) to be repaired, restored, and/or replaced inaccordance with the presently disclosed subject matter. Thus, the term“biocompatible” is meant to refer to a material that can be implantedinternally in a subject as described herein.

The term “composite material”, as used herein, is meant to refer to anymaterial comprising two or more components. One of the components of thematerial can optionally comprise a matrix for carrying cells, such as agel matrix or resin.

As used herein, the phrases “biologically active agent” and“biologically active factor” are used interchangeably and can refer to acompound or mixture of compounds that when added to a cell in cultureinduces the cell to enter differentiation (e.g., differentiate at leastone step further along a pathway of differentiation).

As used herein, the term “effective amount” refers to an amount of abiologically active agent sufficient to produce a measurable response(e.g., a biologically relevant response in a cell exposed to thedifferentiation-inducing agent) in the cell. In some embodiments, aneffective amount of a differentiation-inducing agent is an amountsufficient to cause a precursor cell to differentiate in in vitroculture into a cell of a tissue at predetermined site of treatment. Itis understood that an “effective amount” can vary depending on variousconditions including, but not limited to the stage of differentiation ofthe precursor cell, the origin of the precursor cell, and the cultureconditions.

In some embodiments, an “effective amount” of a “biologically activeagent” can be determined by assaying the ability of different amounts ofa putative biologically active agent to induce the expression of a geneor genes associated with development of a cell that can be used inproviding treatment of a tissue pathology as disclosed herein. Forexample, expression of the gene products aggrecan (for example, thehuman aggrecan gene product disclosed as GENBANK® Accession No. P16112,or a functional fragment or variant thereof) and type II collagen (forexample, the human aggrecan gene product disclosed as GENBANK® AccessionNo. NP_(—)001835, or a functional fragment or variant thereof) areassociated with chondrogenic differentiation. In some embodiments, agene expression level of aggrecan and/or type II collagen is measuredbefore and after a given amount biologically active agent is provided toa culture of ADS cells (for example, hADS cells), and the levels arecompared to determine if the amount of the biologically active agentprovided is an “effective amount”. In some embodiments, the expressionof other genes are similarly determined, including genes that are notassociated with particular cartilaginous tissues including, but notlimited to type I collagen and type X collagen.

The term “expression vector” as used herein refers to a DNA sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, comprising a promoter operatively linked tothe nucleotide sequence of interest which is operatively linked totermination signals. It also typically comprises sequences required forproper translation of the nucleotide sequence. The construct comprisingthe nucleotide sequence of interest can be chimeric. The construct canalso be one that is naturally occurring but has been obtained in arecombinant form useful for heterologous expression.

The term “gene expression” generally refers to the cellular processes bywhich a biologically active polypeptide is produced from a DNA sequenceand exhibits a biological activity in a cell. As such, gene expressioninvolves the processes of transcription and translation, but alsoinvolves post-transcriptional and post-translational processes that caninfluence a biological activity of a gene or gene product. Theseprocesses include, but are not limited to RNA synthesis, processing, andtransport, as well as polypeptide synthesis, transport, andpost-translational modification of polypeptides. Additionally, processesthat affect protein-protein interactions within the cell can also affectgene expression as defined herein.

The terms “heterologous gene”, “heterologous DNA sequence”,“heterologous nucleotide sequence” “exogenous nucleic acid molecule”,and “exogenous DNA segment”, as used herein, refer to a sequence thatoriginates from a source foreign to an intended host cell or, if fromthe same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified, for example bymutagenesis or by isolation from native transcriptional regulatorysequences. The terms also include non-naturally occurring multiplecopies of a naturally occurring nucleotide sequence.

Thus, the terms refer to a DNA segment that is foreign or heterologousto the cell, or homologous to the cell but in a position within the hostcell nucleic acid wherein the element is not ordinarily found. In someembodiments where the heterologous DNA sequence comprises an openreading frame, the heterologous DNA sequence is also referred to as a“transgene”, although the term “transgene” is not limited toheterologous DNA sequences that comprise an open reading frame.

The terms “inhomogeneous”, “inhomogeneity”, “heterogeneous”,“heterogeneity”, and grammatical variations thereof, are meant to referto a scaffold and/or fiber as disclosed herein that does not have ahomogeneous composition along a given length or in a given volumetricsection. In some embodiments, an inhomogeneous tissue engineeringconstruct as disclosed herein comprises a composite material, such as acomposite comprising a three dimensional scaffold as disclosed herein,cells that can develop tissues that substantially provide the functionof bone, cartilage, other joint tissues, or combinations thereof, and amatrix that supports the cells. In some embodiments, an inhomogeneousscaffold as disclosed herein can comprise one or more component systemsthat vary in their properties according to a predetermined profile, suchas a profile associated with the tissue and/or other location in asubject where the scaffold will be implanted. Thus, it is an aspect ofthe terms “inhomogeneous”, “inhomogeneity”, “heterogeneous”,“heterogeneity”, and grammatical variations thereof to encompass thecontrol of individual materials and properties in a scaffold.

The terms “non-linear”, “non-linearity”, and grammatical variationsthereof, refer to a characteristic provided by a scaffold and/or fibersystem as disclosed herein such that the scaffold and/or fiber systemcan vary in response to a strain. As would be appreciated by one ofordinary skill in the art after review of the present disclosure, thescaffolds and/or fiber systems disclosed herein provide stress/stainprofiles that mimic that observed in a target such as predeterminedtissue or joint. As such stress/strain responses are typically describedwith reference to a plot, stress/strain responses can be referred to as“non-linear”. Important non-linear properties of most biological tissuesare significant differences in the strength, stiffness, and/or otherproperties associated with the magnitude of strain, as well assignificant differences in the strength, stiffness, and/or otherproperties as measured in tension as compared to those measured incompression but along the same axis or direction.

When used in the context of a promoter, the term “linked” as used hereinrefers to a physical proximity of promoter elements such that theyfunction together to direct transcription of an operably linkednucleotide sequence.

As used herein, the terms “nucleic acid” and “nucleic acid molecule”mean any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated by the polymerase chain reaction(PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acids can becomposed of monomers that are naturally occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), or analogs of naturallyoccurring nucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Nucleic acids can be eithersingle stranded or double stranded.

The terms “operatively linked” and “operably linked”, as used herein,refer to a promoter region that is connected to a nucleotide sequence(for example, a coding sequence or open reading frame) in such a waythat the transcription of the nucleotide sequence is controlled andregulated by that promoter region. Similarly, a nucleotide sequence issaid to be under the “transcriptional control” of a promoter to which itis operably linked. Techniques for operatively linking a promoter regionto a nucleotide sequence are known in the art.

As used herein, the term “polypeptide” means any polymer comprising anyof the 20 protein amino acids, or amino acid analogs, regardless of itssize or function. Although “protein” is often used in reference torelatively large polypeptides, and “peptide” is often used in referenceto small polypeptides, usage of these terms in the art overlaps andvaries. The term “polypeptide” as used herein refers to peptides,polypeptides and proteins, unless otherwise noted. As used herein, theterms “protein”, “polypeptide” and “peptide” are used interchangeably.The term “polypeptide” encompasses proteins of all functions, includingenzymes.

The term “promoter” or “promoter region” each refers to a nucleotidesequence within a gene that is positioned 5′ to a coding sequence of asame gene and functions to direct transcription of the coding sequence.The promoter region comprises a transcriptional start site, and canadditionally include one or more transcriptional regulatory elements. Insome embodiments, a method of the presently disclosed subject matteremploys a promoter that is active in an endoderm-derived tissue.Exemplary such promoters include promoters that are active in the liver,the pancreas, the spleen, the lung, etc.

A “minimal promoter” is a nucleotide sequence that has the minimalelements required to enable basal level transcription to occur. As such,minimal promoters are not complete promoters but rather are subsequencesof promoters that are capable of directing a basal level oftranscription of a reporter construct in an experimental system. Minimalpromoters include but are not limited to the CMV minimal promoter, theHSV-tk minimal promoter, the simian virus 40 (SV40) minimal promoter,the human β-actin minimal promoter, the human EF2 minimal promoter, theadenovirus E1B minimal promoter, and the heat shock protein (hsp) 70minimal promoter. Minimal promoters are often augmented with one or moretranscriptional regulatory elements to influence the transcription of anoperably linked gene. For example, cell-type-specific or tissue-specifictranscriptional regulatory elements can be added to minimal promoters tocreate recombinant promoters that direct transcription of an operablylinked nucleotide sequence in a cell-type-specific or tissue-specificmanner

Different promoters have different combinations of transcriptionalregulatory elements. Whether or not a gene is expressed in a cell isdependent on a combination of the particular transcriptional regulatoryelements that make up the gene's promoter and the differenttranscription factors that are present within the nucleus of the cell.As such, promoters are often classified as “constitutive”,“tissue-specific”, “cell-type-specific”, or “inducible”, depending ontheir functional activities in vivo or in vitro. For example, aconstitutive promoter is one that is capable of directing transcriptionof a gene in a variety of cell types. Exemplary constitutive promotersinclude the promoters for the following genes which encode certainconstitutive or “housekeeping” functions: hypoxanthine phosphoribosyltransferase (Hprt), dihydrofolate reductase (Dhfr; Scharfmann et al.(1991) Proc Natl Acad Sci USA 88:4626-4630), adenosine deaminase,phosphoglycerate kinase (Pgk), pyruvate kinase, phosphoglycerate mutase,the β-actin promoter (see e.g., Williams et al. (1993) J Clin Invest92:503-508), and other constitutive promoters known to those of skill inthe art. “Tissue-specific” or “cell-type-specific” promoters, on theother hand, direct transcription in some tissues and cell types but areinactive in others.

The terms “replace”, “replacement”, and grammatical variations thereof,refer to any qualitative or quantitative improvement in a target orpredetermined tissue or site of treatment observed upon implantation ofa composition as disclosed herein. For example, these terms are notlimited to full restoration to a normal healthy function, although theseterms can refer to this. Rather, these terms are meant to any level ofimprovement observed in the tissue or at the site.

The terms “reporter gene” and “marker gene” refer to an exogenous geneencoding a product that is readily observed and/or quantitated. Areporter gene is exogenous in that it originates from a source foreignto an intended host cell or, if from the same source, is modified fromits original form. Non-limiting examples of detectable reporter genesthat can be operatively linked to a transcriptional regulatory regioncan be found in Alam and Cook (1990) Anal Biochem 188:245-254, and PCTInternational Publication No. WO 97/47763. Exemplary reporter genesinclude the lacZ gene (see e.g., Rose and Botstein (1983) MethodsEnzymol 101:167-180), Green Fluorescent Protein (GFP; Cubitt et al.(1995) Trends Biochem Sci 20:448-455), luciferase, and chloramphenicolacetyl transferase (CAT). Any suitable reporter and detection method canbe used, and it will be appreciated by one of skill in the art that noparticular choice is essential to or a limitation of the presentlydisclosed subject matter.

The terms “resin”, “matrix”, or “gel” are used the art-recognized senseand refer to any natural or synthetic solid, liquid, and/or colloidalmaterial that has characteristics suitable for use in accordance withthe presently disclosed subject matter. Representative “resin”,“matrix”, or “gel” materials thus comprise biocompatible materials. Insome embodiments, the “resin”, “matrix”, or “gel” can occupy the porespace of a fiber scaffold as disclosed herein.

The terms “restore”, “restoration”, and grammatical variations thereofrefer to any qualitative or quantitative improvement in a target orpredetermined tissue or and/or site of treatment observed uponimplantation of a composition as disclosed herein. Thus, these terms arenot limited to full restoration of the tissue and/or site to a normalhealthy function, although these terms can refer to this. Rather, theseterms are meant to refer to any measurable and/or observable level ofimprovement in the tissue and/or site.

The terms “resurface”, “resurfacing”, and grammatical variations thereofrefer to any qualitative or quantitative replacement of least themajority of the surface area of the surface of tissue upon implantationof a composition as disclosed herein. These terms can also refer to anydesired depth of resurfacing; such as but not limited to a layer ofmicron thickness, to multiple layers of tissue including multiple tissuetypes, and/or to replacement of a complete structure that provides asurface at the site of treatment. Thus, these terms are not limited tofull replacement of the tissue and/or site, although these terms canrefer to this. Rather, these terms are meant to refer to replacement ofany fraction of the native tissue beyond what is considered by oneskilled in the art as a “focal defect”. A representative surface is anarticulating surface of a joint.

As used herein, the term “selectable marker” refers to a gene or geneproduct that confers a growth advantage to a cell that expresses it. Forexample, a selectable marker can allow a cell that expresses it to growin the presence of a chemical (e.g., a drug such as G418) that wouldinhibit the growth of or kill cells that do not express the selectablemarker. Selectable marker genes include, but are not limited toantibiotic resistance genes, for example the antibiotic resistance geneconfers neomycin resistance (herein referred to as the “neo gene”).

The term “transcriptional regulatory sequence” or “transcriptionalregulatory element”, as used herein, each refers to a nucleotidesequence within the promoter region that enables responsiveness to aregulatory transcription factor. Responsiveness can encompass a decreaseor an increase in transcriptional output and is mediated by binding ofthe transcription factor to the DNA molecule comprising thetranscriptional regulatory element.

The term “transcription factor” generally refers to a protein thatmodulates gene expression by interaction with the transcriptionalregulatory element and cellular components for transcription, includingRNA Polymerase, Transcription Associated Factors (TAFs),chromatin-remodeling proteins, and any other relevant protein thatimpacts gene transcription.

The terms “viscoelastic”, “viscoelasticity”, and grammatical variationsthereof, are meant to refer to a characteristic provided by a scaffoldand/or fiber system as disclosed herein that can vary with a time and/orrate of loading. It is thus envisioned that appropriately viscoelasticscaffolds and/or fiber systems provide time and/or rate of loadingcharacteristics that match or approximate that observed in thepredetermined tissue or site. This characteristic pertains todissipation of energy, which can be provided by the scaffold itselfand/or by the scaffold as a composite with cells growing therein, andcan also be accomplished by virtue of the choices of fibers that areincluded in the scaffold. As a particular example, it can be desirableto provide a scaffold that approximates the viscoelastic properties ofcartilage.

All references cited in the specification, including patents, patentapplication publications, journal articles, and all other referencescited herein, are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

II. Cells and Reagents

II.A. Representative Cells for Joint Resurfacing

The presently disclosed implantable composition can comprise one or morecells that can develop into a suitable replacement of a target tissue(e.g., bone, cartilage, or both bone and cartilage). Particularly, theone or more cells comprise, or are derived from, a precursor cells, suchas but not limited to a stem cell. As used herein, the term “stem cell”refers to any unipotent, multipotent, pluripotent and/or totipotent cellthat can be differentiated into a desired lineage. As such, thepresently disclosed subject matter can employ stem cells that can bedifferentiated into a tissue appropriate for replacement of nativepathological tissues. Representative stem cells include embryonic stem(ES) cells, embryonic germ (EG) cells (e.g., pluripotent cells derivedfrom primordial germ cells), and somatic stem cells (alternativelyreferred to herein as “adult stem cells”).

In some embodiments, the one or more cells described herein comprise anadult stem cell. Adult stem cells can be derived from various adulttissues including, but not limited to liver, bone marrow, umbilical cordblood, brain, peripheral blood, blood vessels, skeletal muscle, adiposetissue, and skin. Methods for the isolation, culturing, and manipulationof adult stem cells from various sources can be found in U.S. Pat. Nos.6,242,252 and 6,872,389 (hepatic stem cells); U.S. Pat. No. 6,387,367(hematopoietic/mesenchymal stem cells); Kogler et al. (2004) J Exp Med200:123-135 (placental cord blood); Williams et al. (1999) The AmericanSurgeon 65:22-26 (skeletal muscle); U.S. Pat. No. 6,777,231 (adiposetissue); and Blanpain et al. (2004) Cell 118:635-648 (skin), the entirecontents of each of which are hereby incorporated in their entireties.

Representative techniques for deriving, growing, and manipulating EScells and EG cells are disclosed in the following publications: Evansand Kaufman (1981) Nature 292:154-156; Martin (1981) Proc Natl Acad SciUSA 78:7634+7638; Robertson (1986) Trends Genet 2:9-13; PCTInternational Patent Application Publications WO 96/22362; WO 97/32033;and WO 98/43679; and U.S. Pat. Nos. 6,200,806; 6,090,622; 5,843,780;5,690,926; 5,670,372; and 5,453,357; and references therein, all ofwhich are incorporated by reference herein in their entireties.

Thus, the presently disclosed subject matter provides in someembodiments the use of the cells described herein for treatment of jointdisease. Currently, there are limited treatment options forosteoarthritis, as one example of joint disease. For advanceddegeneration, the only current treatment option is replacement of thejoint with artificial materials, which include polymers and metals,which effectively act as an artificial joint. While the jointreplacement surgeries alleviate pain and restore some function in manyof the patients in the short-term, these joint replacements are notintended for long-term use and often require difficult surgicalrevisions, potentially leading to significant post-operativecomplications. Some post-operative complications associated with the useof artificial materials include device related osteopenia, osteolysis,excessive wear of the bearing surfaces of the artificial device, andfracture of the bones supporting the implant. Disclosed herein for thefirst time are approaches for the complete resurfacing of the diseasedarticular surface with a bioartificial implant, which avoids thecomplications associated with the introduction of artificial materialsdue to the biologic nature of the composition of the implantedstructure. Further disclosed herein is the use of progenitor, stem, orprimary cells in conjunction with a composition that comprises a mediumcapable of supporting the growth and differentiation of the cells intofunctional tissue, but not necessarily recapitulating the nativestructure of the tissue.

II.B. Adipose Derived Stem (ADS) Cells

The ADS and ADS-derived cells of an aspect of the presently disclosedsubject matter are useful in providing a source of differentiated andfunctional cells for research, transplantation, and development oftissue engineering products for the treatment of mammalian disease andtraumatic injury repair. Thus, in some aspects, the presently disclosedsubject matter provides methods for differentiating ADS cells comprisingculturing the cells in a composition that comprises a medium capable ofsupporting the growth and differentiation of the cells. The presentlydisclosed subject matter further provides methods for the introductionof these cells into tissue defect areas in need of repair. In someembodiments the tissue defect areas can be treated by exclusively usingthe ADS and ADS-derived cells of the presently disclosed subject matter.

As an example of one tissue pathology, there are currently limitedtreatment options for focal cartilage lesions. One treatment optioninvolves drilling into the subchondral bone and exposing the cartilagetissue to growth factors and other molecular agents from the vascularsupply found in the bone in the hope that regeneration of the cartilagelesion occurs. A second technique involves the transfer of “healthy”cartilage from non-load bearing areas to “unhealthy” areas to replacethe degenerated cartilage. Thirdly, a cell-based therapy exists thatutilizes ex vivo cultured autologous chondrocytes reimplanted at thedefect site to regenerate the damaged tissue.

All three of these techniques are marked by varying degrees of success,and accordingly, novel techniques and methodologies are needed for theeffective remodeling, repair, regrowth, and/or regeneration of cartilagelesions. The presently disclosed subject matter relates to replacementof damaged cartilage as well as other tissues and has broad applicationsin the field of tissue engineering and regenerative medicine.

ADS cells provide a readily accessible, abundant source of multipotentprogenitor cells for applications in tissue engineering and othercell-based therapies. In particular, the potential use of ADS cells forthe remodeling, repair, regrowth, and/or regeneration of cartilage hasbeen explored. However, employing the chondrogenic differentiationtechniques currently available in the art only results in a mildchondrogenic phenotype in in vitro culture. On the contrary, disclosedherein for the first time are approaches for the unambiguous and robustdifferentiation of ADS cells along a lineage appropriate forreplacement/regeneration of pathological tissue such as degenerated,injured, or damaged cartilage or other connective tissue, and use ofthese cells in conjunction with a composition that comprises a mediumcapable of supporting the growth and differentiation of ADS cells intofunctional tissue, but not necessarily recapitulating the nativestructure of the tissue.

Thus, the presently disclosed subject matter provides in someembodiments methods and systems for inducing specific phenotypes in ADScells for the treatment of various tissue pathologies. In contrast tocurrent technologies that are compromised by difficulties in obtainingappropriate stem cells and in differentiating the stem cells as desiredin culture, the presently disclosed subject matter provides methods andsystems for promoting ADS cell differentiation at a significantlyincreased rate over previously known methods.

In some embodiments of the presently disclosed subject matter, methodsand systems are provided for inducing differentiation comprisingproviding to ADS cells in culture an effective amount of a biologicallyactive factor (e.g., BMP-6) or a functional fragment thereof.

In some embodiments of the presently disclosed subject matter, methodsand systems are provided for determining whether a cell hasdifferentiated into a desired phenotype. Particularly, because the cellsof the presently disclosed subject matter have a specific phenotype,they can be employed in tissue engineering. In this regard, thepresently disclosed subject matter provides in some embodiments methodsof maintaining the ADS cells under conditions sufficient for them toexpand and differentiate to form the desired subject matter.

II.C. Isolation of ADS Cells

ADS cells (e.g., hADS cells) are isolated from a subject or obtaineddirectly from an established cell culture line. The subject can be aliveor dead, so long as the ADS cells within the subject are viable.Typically, ADS cells are obtained from living donors, usingwell-recognized protocols such as surgical or suction lipectomy. Suchcells can be isolated from the subject to be treated, or from a subjectdifferent from the subject to be treated. In some embodiments, thesubject from which the cells are isolated is of a different species thanthe subject into which the cells are to be transferred. Thus, in someembodiments, the ADS cells can be derived from the adipose tissue of aprimate, a higher primate (e.g., baboon or ape), or from human adiposetissue, using the methods described herein.

Thus, in some embodiments, the ADS cells are syngeneic (also referred toherein as “autologous”) to the subject into which the ADS cells and/orADS-derived cells are intended to be placed, and in some embodiments theone or more cells are allogeneic (also referred to herein as“heterologous”) to the subject. In those embodiments where the one ormore cells are allogeneic or xenogeneic to the subject, the subject canbe treated as necessary with immunosuppressant drugs such ascyclosporin, azathioprines, or corticosteroids using well-knowntechniques. Representative immunosuppressive drugs also include, but arenot limited to, basiliximab (SIMULECT®; available from NovartisPharmaceuticals Corp., East Hanover, N.J., United States of America),daclizumab (ZENAPAX®, available from Hoffmann-La Roche Inc., Nutley,N.J., United States of America), muromonab CD3 (ORTHOCLONE OKT3®,available from Ortho Biotech Products, L.P., Bridgewater, N.J., UnitedStates of America) and tacrolimus (PROGRAF®, available from AstellasPharma US, Inc., Deerfield, Ill., United States of America).

As would be readily understood by one of skill in the art, ADS cellsrefer to stem cells that originate from adipose tissue and are capableof self-renewal. By “adipose” is meant any fat tissue. ADS cells can beisolated from any source of adipose tissue in the subject, although insome embodiments, the ADS cells are isolated from an adipose depot inthe body selected from the group consisting of the subcutaneous abdomen,the thigh, the buttocks, and the infrapatellar fat pad. Adipocytes canbe harvested by liposuction on an outpatient basis, a relativelynon-invasive procedure with cosmetic effects that are acceptable to thevast majority of patients. It is well documented that adipocytes are areplenishable cell population. Even after surgical removal byliposuction or other procedures, it is common to see a recurrence ofadipocytes in an individual over time.

ADS cells can comprise a primary cell culture or an immortalized cellline. While stem cells represent less than 0.01% of the bone marrow'snucleated cell population, there are up to 8.6×10⁴ stem cells per gramof adipose tissue (Sen, et al. (2001) Journal of Cellular Biochemistry,81:312-319). Ex vivo expansion over 2 to 4 weeks yields up to 500million stem cells from 0.5 kilograms of adipose tissue. These cells canbe used immediately or cryopreserved for future autologous or allogeneicapplications.

In addition, the isolated ADS cells can be further separated intosubpopulations of ADS cells based upon any observable, quantifiable, orother trait of the cells for which a separation technique is availableor can be designed. In some embodiments, isolated ADS cells areseparated into subpopulations using fluorescence-activated cell sorting(FACS) based on the appearance of one or more of cell surface markers.In some embodiments, the following cell surface markers can be employedfor separating ADS cells into subpopulations: CD10, CD13, CD31, CD34,CD36, CD44, CD49, CD54, CD55, CD59, CD65 CD105, and CD166.

In some embodiments, the isolated ADS cells can be separated intosubpopulations of ADS cells based on differential expression of variousgenes. In some embodiments, isolated ADS cells are separated intosubpopulations based on differential expression of aldehydedehydrogenase (ALDH), various members of the TGF-β superfamily, TGF-βsuperfamily receptor, and/or telomerase activity. In some embodiments,the isolated ADS cells are separated into subpopulations of ADS cellsbased on telomere length. In some embodiments, the isolated ADS cellscan be separated into subpopulations of ADS cells based on efflux ofmacromolecules including, but not limited to dyes (e.g., Hoechst 33342)or nucleic acid labels. In some embodiments, the isolated ADS cells canbe separated into subpopulations of ADS cells based on responsiveness toa particular growth factor (e.g., BMP-6). It is understood that two ormore of these separation strategies can be employed together to producesubpopulations of ADS cells either before or after the induction ofdifferentiation.

Such isolated ADS cells and populations can be clonally expanded, ifdesired, using a suitable method for cloning cell populations. Forexample, a proliferated population of cells can be physically picked andseeded into a separate plate (or the well of a multi-well plate).Alternatively, the cells can be subcloned onto a multi-well plate at astatistical ratio for facilitating placing a single cell into each well(e.g., from about 0.1 to about 1 cell/well). In some embodiments, thecells can be cloned by plating them at low density (e.g., in apetri-dish or other suitable substrate) and isolating them from othercells using devices such as a cloning rings. Alternatively, where anirradiation source is available, clones can be obtained by permittingthe cells to grow into a monolayer and then shielding one andirradiating the rest of cells within the monolayer. The surviving cellsthen can grow into a clonal population.

II.D. Genetic Manipulation of Cells

In some embodiments, the cells, for example ADS cells, can begenetically modified, e.g., to express exogenous genes or to repress theexpression of endogenous genes. In some embodiments, the presentlydisclosed subject matter provides methods of genetically modifying suchcells and populations. In accordance with these methods, the cells canbe exposed to an expression construct comprising a nucleic acidincluding a transgene, such that the nucleic acid is introduced into thecell under conditions appropriate for the transgene to be expressedwithin the cell. The transgene generally is an expression cassette,including a coding polynucleotide operably linked to a suitablepromoter. The coding polynucleotide can encode a protein, or it canencode a biologically active (e.g., functional) fragment of a protein.

Thus, for example, the coding polynucleotide can encode a geneconferring resistance to a toxin, a hormone (such as peptide growthhormones, hormone releasing factors, sex hormones, adrenocorticotrophichormones, cytokines (e.g., interferins, interleukins, lymphokines),etc.), a cell-surface-bound intracellular signaling moiety (e.g., celladhesion molecules, hormone receptors, etc.), a factor promoting a givenlineage of differentiation, etc. Of course, where it is desired toemploy gene transfer technology to deliver a given transgene, thesequence will be known. In some embodiments, the coding polynucleotideencodes a growth factor. In some embodiments, the coding polynucleotideencodes BMP-6 or a functional fragment thereof. In some embodiments, thecoding polynucleotide encodes BMP-6 receptor or a functional fragmentthereof.

The cells can be stably or transiently transfected or transduced with anucleic acid of interest using a plasmid, viral or alternative vectorstrategy. With respect to ADS cells, nucleic acids of interest include,but are not limited to, those encoding gene products which enhance theproduction of extracellular matrix components found in cartilage;examples include, collagen type II, TGF-β, BMP, activin and insulin-likegrowth factor.

Thus, in some embodiments, the transduction of regulatory genes into thecells, for example ADS cells, can be performed with viral vectors(adenovirus, retrovirus, adeno-associated virus, or other vector)purified by cesium chloride banding or any other well-known method at amultiplicity of infection (viral units:cell) of between 10:1 to 2000:1.Cells can be exposed to the virus in serum-free or serum-containingmedium in the absence or presence of a cationic detergent such aspolyethyleneimine or Lipofectamine™ (available from Invitrogen,Carlsbad, Calif., United States of America) for a period of 1 hour to 24hours (Byk et al. (1998) Human Gene Therapy 9:2493-2502; Sommer et al.(1999) Calcif. Tissue Int. 64:45-49) or in three dimensional cultures byincorporation of the plasmid DNA vectors directly into a biocompatiblepolymer (Bonadio et al. (1999) Nat. Med. 5:753-759).

In some embodiments, cells, for example ADS cells, are transfected withthe gene to be expressed to produce cells having stably incorporatedtherein the DNA encoding the molecules to be expressed. Stabletransfections can be obtained by culturing and selecting for expressionof the desired encoded molecules. In some embodiments, the cells thatexhibit stable expression can be seeded onto or into the appropriatefiber matrix and implanted in a subject. For the tracking and detectionof functional proteins encoded by these genes, the viral or plasmid DNAvectors can contain a readily detectable marker gene, such as the greenfluorescent protein (GFP) or β-galactosidase enzyme, both of which canbe tracked by histochemical means.

Within the expression cassette, the coding polynucleotide can beoperably linked to a suitable promoter. Examples of suitable promotersinclude prokaryotic promoters and viral promoters (e.g., retroviralinverted terminal repeats (ITRs), long terminal repeats (LTRs),immediate early viral promoters (IEp), such as herpes virus IEp (e.g.,ICP4-IEp and ICP0-IEp), cytomegalovirus (CMV) IEp, and other viralpromoters, such as Rous Sarcoma Virus (RSV) promoters, and MurineLeukemia Virus (MLV) promoters). Other suitable promoters are eukaryoticpromoters, such as enhancers (e.g., the rabbit β-globin regulatoryelements), constitutively active promoters (e.g., the β-actin promoter,etc.), signal specific promoters (e.g., inducible promoters such as apromoter responsive to RU486, etc.), and tissue-specific promoters. Itis well within the skill of the art to select a promoter suitable fordriving gene expression in a predefined cellular context. The expressioncassette can include more than one coding polynucleotide, and it caninclude other elements (e.g., polyadenylation sequences, sequencesencoding a membrane-insertion signal or a secretion leader, ribosomeentry sequences, transcriptional regulatory elements (e.g., enhancers,silencers, etc.), and the like), as desired.

The expression cassette containing the transgene can be incorporatedinto a genetic vector suitable for delivering the transgene to thecells. Depending on the desired end application, any such vector can beso employed to genetically modify the cells (e.g., plasmids, naked DNA,viruses such as adenovirus, adeno-associated virus, herpes viruses,lentiviruses, papillomaviruses, retroviruses, etc.). Any method ofconstructing the desired expression cassette within such vectors can beemployed, many of which are well known in the art (e.g., direct cloning,homologous recombination, etc.). Of course, the choice of vector willlargely determine the method used to introduce the vector into the cells(e.g., by protoplast fusion, calcium-phosphate precipitation, gene gun,electroporation, infection with viral vectors, etc.), which aregenerally known in the art.

In some embodiments, the genetically altered (e.g., ADS) cells can beemployed as bioreactors for producing the product of the transgene. Insome embodiments, the genetically modified cells are employed to deliverthe transgene and its product to a subject. For example, the cells, oncegenetically modified, can be introduced into the subject underconditions sufficient for the transgene to be expressed in vivo.

II.E. Induction of Differentiation for Tissue Replacement

Another object of the presently disclosed subject matter is to providefor the identification and study of compounds that enhance thedifferentiation of ADS cells (e.g., hADS cells) into cells capable offorming an extracellular matrix capable of functioning in place of thenative tissue. Representative, non-limiting extracellular matrixproteins are disclosed in the Examples. Compounds that enhancedifferentiation can be of value in the treatment of partial or fullcartilage defects, osteoarthritis, traumatized cartilage, and cosmeticsurgery of inborn defects including cleft palate and deviated septum,among other treatments.

After isolation, ADS cells can be cultured in vitro for a time and underconditions sufficient to induce one or more of the cells to undergodifferentiation. In some embodiments, one or more biologically activeagents are added to the culture medium. In some embodiments, abiologically active agent comprises a BMP-6 polypeptide, or a functionalfragment thereof. In some embodiments, a biologically active agentcomprises a BMP-6 polypeptide, or a functional fragment thereof, incombination with one or more additional growth factors and/or cytokines.It is understood that for any polypeptides that are employed asconstituents of a biologically active agent, it is not necessary thatfull length polypeptides be employed, as functional fragments can alsobe used. As used herein, the term “functional fragment” refers to asubsequence of a polypeptide (or a subsequence of a nucleic acidencoding such a polypeptide fragment) that is characterized by at leastsome activity in differentiation when part of a biologically activeagent.

In some embodiments, the method of inducing differentiation in stemcells comprises (a) providing to a stem cell in culture an effectiveamount of a biologically active factor (e.g., BMP-6) or a functionalfragment thereof; and (b) growing the stem cell (e.g. ADS cell) inculture for a time sufficient for differentiation to occur, whereindifferentiation is determined to have occurred when at least one stemcell (e.g. ADS cell) exhibits expression of a macromolecule associatedwith the native, healthy tissue to be replaced (i.e., nativeextracellular composition of the tissue prior to injury, disease, ordegeneration). Representative, non-limiting extracellular matrixproteins are disclosed in the Examples.

In some embodiments, the method further comprises passaging the stemcell (e.g. ADS cell) repeatedly (e.g., at least twice) in culture,wherein the passaging enhances an ability of the cell to express atleast one macromolecule associated with the predetermined target tissueupon exposure to a biologically active factor or a functional fragmentthereof. Representative macromolecules are disclosed in the Examples.

In some embodiments, the method further comprises providing to the stemcells (e.g. ADS cells) a predetermined effective amount of a secondbiologically active factor.

Representative biologically active factors can be selected from thegroup including, but not limited to, growth factors, e.g., TGF-βsuperfamily members (including but not limited to BMP-6, TGF-β1, TGF-β2,TGF-β3), IGF-1, FGF, an EGF, a PDGF, a parathyroid hormone relatedpeptide (PTHrP), an interleukin, cytokines, chemokines, gelatins,laminins, fibronectins, thrombins, lipids, cartilage oligomeric proteins(COMP), thrombospondins, fibrins, fibrinogens, Matrix-GLA protein,chondrocalcin, tenascin, a mineral, an RGD peptide, an RGD-peptidecontaining molecule, elastin, hyaluronic acid, a glycosaminoglycan, aproteoglycan and other molecules that alone or in combination arecapable of inducing the differentiation of an ADS cell into a cell or acell that is further differentiated along the appropriate target lineagethan is the ADS cell. In some embodiments a composition to beadministered to cells can consist essentially of a given biologicallyactive agent (such as but not limited BMP-6), and in some embodimentssuch a composition can consist of a given biologically active agent.

In some embodiments, the biologically active agent is selected from thegroup including but not limited to parathyroid hormone, a transforminggrowth factor (e.g., a TGF-α and/or a TGF-β), an insulin-like growthfactor (e.g., IGF-I), a platelet-derived growth factor (PDGF), afibroblast growth factor (FGF), an epidermal growth factor (EGF), avascular endothelial growth factor (VEGF), and combinations thereof. Insome embodiments, the biologically active agent is a bone morphogeneticprotein (BMP), and in some embodiments the BMP is BMP-6 (e.g., humanBMP-6). In some embodiments, the human BMP-6 comprises amino acids374-513 of GENBANK® Accession No. NP_(—)001709, or a functional fragmentor variant thereof, and/or is encoded by a nucleic acid comprisingGENBANK®Accession No. NM_(—)001718, or a function fragment or variantthereof.

Further, it is to be understood that in some embodiments, thebiologically active agent (e.g., BMP-6) can be incorporated into animplantable composition of the presently disclosed subject matter forcontrolled release over time.

II.F. Bone Morphogenic Proteins

Bone morphogenetic proteins (BMPs) are characterized by their ability topromote, stimulate or otherwise induce the formation of cartilage and/orbone. Accordingly, members of the BMP family of proteins can be used incompositions to induce bone and/or cartilage formation, wound healingand tissue repair, treatment of bone and/or cartilage defects,periodontal disease and other tooth repair processes, treatment ofosteoporosis and increase of neuronal survival.

It has been presently discovered, as discussed herein in detail, thatBMP-6 in combination with several growth factors, supplements, and othersoluble mediators increases the gene expression and biosynthesis ofcollagen and proteoglycan by several orders of magnitude as compared toother differentiation protocols that have been previously published.Further, it has also been shown herein that BMP-6 in the absence ofother growth factors strongly promotes a robust differentiation towardsa generated tissue type which may be used to replace many differenttissue types (e.g., ligament, tendon, IVD, meniscus, and cartilage).BMP-6 is a member of a different BMP family subgroup than are BMP-2 andBMP4, and is thus different from BMP-1, -2,-3,-4, and -5. In someembodiments, the effective amount of BMP-6 ranges from about 1picogram/mL to about 10 milligram/mL.

II.G. Detection of Differentiation

After culturing the cells in a suitable medium for a suitable time(e.g., several days to a week or more), the cells can be assayed todetermine whether, in fact, they have differentiated to acquire physicalqualities of a desired cell type. Thus, in some embodiments thepresently disclosed subject matter provides methods for testing ADScell-derived cells.

One measurement of differentiation per se is telomere length,undifferentiated stem cells having longer telomeres than differentiatedcells; thus, the cells can be assayed for the level of telomeraseactivity. Alternatively, RNA or proteins can be extracted from the cellsand assayed (via Northern hybridization, rtPCR, Western blot analysis,etc.) for the presence of markers indicative of the desired phenotype.

Thus, methods for determining whether an ADS cell has differentiatedinto a desired phenotype are also provided. In some embodiments, themethods comprise (a) obtaining mRNA from an ADS cell that has beenexposed to a biologically active agent; and (b) determining from themRNA a level of expression of at least one gene associated with adesired differentiation, wherein the level of expression determined forthe at least one gene is indicative of differentiation of the ADS cell.In some embodiments, the methods comprise determining from the ADS cella level of expression of at least one protein associated with a desireddifferentiation. In some embodiments, the cells can be assayedimmunohistochemically or stained, using tissue-specific stains.

In some embodiments, the methods comprise measuring expression of atleast one gene associated with a desired differentiation to determinewhen at least a subpopulation of the ADS cells have differentiated. Insome embodiments, the appearance of an ADS cell-derived phenotype istested and/or confirmed by testing the ADS cell grown in culture todetermine when differentiation has occurred, wherein the testingcomprises utilizing a technique for measuring gene expression todetermine when the ADS cell has differentiated, and wherein thetechnique for measuring gene expression comprises measuring a level ofexpression of at least one gene associated with a desired phenotype. Insome embodiments, the at least one gene associated with a desireddifferentiation is selected from the group consisting of aggrecan, typeI collagen, type II collagen, type X collagen, and combinations thereof.

Accordingly, the presently disclosed subject matter provides methods foridentifying cells, for example ADS-derived cells, suitable for use intherapeutic applications. In some embodiments, the differentiated ADScell is identified as suitable for use in therapeutic restorative andregenerative techniques when gene expression measurements, proteinmeasurements, or combinations thereof meet predetermined parameters,such as but not limited to those disclosed in the Examples presentedherein.

Other methods of assessing developmental phenotype are known in the art,and any of them are appropriate. For example, the cells can be sorted bysize and granularity. Also, the cells can be used to generate monoclonalantibodies, which can then be employed to assess whether theypreferentially bind to a given cell type. Correlation of antigenicitycan confirm that the stem cell has differentiated along a givendevelopmental pathway.

II.H. Administration of Cells to Subjects

As is known in the art, when the cells are to be administered to asubject, it is preferable that the cell culture medium be biologicallycompatible with the subject. Stated another way, in some embodiments thecell culture medium that is used to culture and induce cells does notcontain any components that would be expected to negatively affect thehealth of the subject after administration of the induced cells. Thus,an appropriate cell culture medium can also comprise one or moreserum-free medium supplements. As used herein, the term “serum-freemedium supplement” refers to a supplement that can be added to a mediumto replace some or all of the serum that would normally be added to themedium to support the propagation and/or maintenance of cells inculture.

Serum-free medium supplements typically comprise about 10-25 mM HEPES,about 1-4 grams per liter (g/L) sodium bicarbonate, up to about 5micrograms per liter (μg/L) hypoxanthine, up to about 10 μg/L thymidine,up to about 1.5 g/L sodium pyruvate, up to about 2.0 g/L L-glutamine,and up to about 30 μg/L phenol red. Various trace elements and othergrowth factors can also be added to serum-free medium supplements. Anexemplary serum-free medium supplement is OPTI-MEM® I reduced serummedium supplement, sold by Invitrogen Corp. (Carlsbad, Calif., UnitedStates of America) in powder and liquid forms (Catalog Nos. 22600-050,22600-134, 11058-021, 31985-062, 31985-070, 31985-088, and 51985-034).Other serum-free medium supplements include BIOGRO-1 and BIOGRO-2(Biological Industries Ltd., Kibbutz Beit Haemek, Israel) and theNutridoma family of serum free medium supplements sold by Roche AppliedScience (Indianapolis, Ind., United States of America).

For analysis and/or administration into a subject, the induced cells canbe treated with trypsin/EDTA in order to form a single cell suspensionand resuspended in an appropriate pharmaceutically acceptable carriersuch as phosphate-buffered saline. Representative carriers include, butare not limited to, calcium alginate, agarose, types I, II, IV or othercollagen isoform, fibrin, poly-lactic/poly-glycolic acid, hyaluronatederivatives or other materials (Perka et al. (2000) J. Biomed. Mater.Res. 49:305 311; Sechriest et al. (2000) J. Biomed. Mater. Res. 49:534541; Chu et al: (1995) J. Biomed. Mater. Res. 29:1147 1154; Hendricksonet al. (1994) Orthop. Res. 12:485 497). Thus, in some embodiments, theinduced cells can be transplanted into a desired site in a subject(i.e., a joint) to promote in situ repair or regeneration of cartilage,bone, or cartilage and bone. In some embodiments, two different types ofcells are administered, for example, another cell type is administeredalong with a progenitor, stem, or primary cell. The other cell type canbe selected from the group consisting of, but not limited to, achondrocyte, a fibroblast, an osteoblast, a myoblast, a neuron, aprogenitor cell, and combinations thereof.

Thus, in some embodiments the cells can be administered to a targettissue selected from the group consisting of, but not limited to,articular cartilage, non-articular cartilage, auricular cartilage,tracheal cartilage, laryngeal cartilage, nasal cartilage, growth platecartilage, meniscus, labrum, and intervertebral disc. In someembodiments, the target tissue can comprise a musculoskeletal or dentalconnective tissue selected from the group consisting of, but not limitedto, a tendon, ligament, periodontal ligament, fascia, and muscle. It isto be understood that the target tissue can comprise multiple tissuetypes that are integrated with one another, selected from the groupconsisting of, but not limited to, bone and cartilage (e.g., anosteochondral junction), muscle and tendon (e.g., a myotendinousjunction), and ligament and bone (e.g., an insertion site).

In some embodiments, the target tissue is the surface of an articulatingjoint it is to be understood that the target tissue can comprisemultiple tissue types that are integrated with one another, selectedfrom the group consisting of, but not limited to, bone and cartilage(e.g., an osteochondral junction), muscle and tendon (e.g., amyotendinous junction), and ligament and bone (e.g., an insertion site).

In some embodiments, the one or more cells to be administered to asubject are present in a matrix (e.g., a gel) within the pores of afiber scaffold. The fiber scaffold can be used as a substrate tofacilitate the growth and/or differentiation of cells. Thus, in someembodiments, the cells can be used to grow pieces of functionalcartilage and/or bone in vitro for implantation into a desired site inthe patient (e.g., site of pathology or joint surface).

III. Scaffolds for Implantable Compositions

The presently disclosed subject matter provides in some embodimentsimplantable compositions comprising scaffolds for use at a predeterminedsite in a subject. In some embodiments the presently disclosed subjectmatter provides a joint replacement or resurfacing implant (for example,an implant that is intended to cover the majority of the articulatingsurface or surfaces of a joint) adapted for use with a predeterminedjoint that combines novel composite biomaterials with or without cellsto produce a composite implant comprising cells that differentiate into,and/or have differentiated to, tissues capable of substantial boneand/or cartilage function, among other functions. In contrast to currenttechnologies that seek to repair small pieces of cartilage or focaldefects in a predetermined joint, a joint replacement implant of thepresently disclosed subject matter is used in some embodiments toresurface the majority or entirety of surfaces of damaged or diseasedjoints.

A representative joint addressed in some embodiments of the presentlydisclosed subject matter is the hip, due to the high incidence of hiposteoarthritis. In some embodiments of the presently disclosed subjectmatter, a joint replacement implant is engineered into a hemisphericalshape for use in a hip replacement. In some embodiments, the jointreplacement implant is grown using human adult stem, other progenitor,and or primary cells seeded onto three-dimensional woven compositebiomaterial matrices that provide desired biomechanical properties. Acombination of growth modulating materials as defined herein andphysical stimuli can be used within a bioreactor to promotedifferentiation of integrated bone and cartilage within the jointreplacement implant. This approach can be used to fabricate implants,which can provide substantial cartilage or cartilage/bone function forreplacement of the surfaces of the joint (e.g., femoral head and/oracetabular cup), shoulder, knee, finger (e.g., thumb, phalanges,carpo-metacarpal, trapeziometacarpal), temporomandibular joint, patella,elbow, ankle, or any other diarthrodial joints.

A representative, non-limiting process that can be employed to form anexemplary joint replacement implant, a bioartificial hip, isschematically depicted in FIG. 8. As shown in FIG. 8, liposuction can beemployed to isolate autologous (or heterologous) stem cells (includingbut not limited to adipose-derived stem cells), which can then beexpanded in culture. Once a sufficient number of such stem cells areproduced, the cells can be concentrated and suspended in a matrix suchas a gel biomaterial and applied to a three-dimensional scaffold, whichis then placed in culture or in a bioreactor until sufficient growthand/or differentiation has occurred so that the resulting jointreplacement implant can be implanted into the subject.

Thus, the presently disclosed subject matter represents a significantdeparture from previous approaches in that a living tissue substitutefor the entire joint surface is provided, rather than a repair of anisolated defect. Since the joint replacement implant comprises livingtissue, it can integrate with the subject's tissues and can requiresignificantly less invasive surgery and minimal removal of nativetissues. In some embodiments, a joint replacement implant is providedthat can be implanted using minimally invasive surgery as a temporaryreplacement for an osteoarthritic hip or other joint surfaces. In suchembodiments, standard prosthetic joint replacement surgery can bedelayed significantly, such as by 5-10 years or longer. This can be ofinterest in to certain subjects, including younger and/or activesubjects.

III.A. Fibers

Disclosed herein, in some embodiments, are joint replacement implantscomprising a fiber scaffold. Two-dimensional or three-dimensional fiberscaffolds can be employed. These scaffolds can comprise systems offibers, wherein, for example for a three-dimensional fiber system, twoof the three fiber systems define an upper layer, a lower layer, and amedial layer between the upper layer and the lower layer within thethree-dimensional fiber scaffold, and wherein one of the at least threefiber systems interconnects the upper layer, the lower layer, and themedial layer. The at least three fiber systems can each comprise abiocompatible material, and the biocompatible material can comprise anabsorbable material, a non-absorbable material, or combinations thereof.

Fibers can be monofilament, multifilament, or a combination thereof, andcan be of any shape or cross-section including, but not limited tobracket-shaped (i.e., [), polygonal, square, I-beam, inverted T shaped,or other suitable shape or cross-section. The cross-section can varyalong the length of fiber. Fibers can also be hollow to serve as acarrier for macromolecules (e.g., antibiotics, growth factors, etc.),cells, and/or other materials as described herein. In some embodiments,the fibers can serve as a degradable or non-degradable carriers todeliver a specific sequence of growth factors, antibiotics, orcytokines, etc., embedded within the fiber material, attached to thefiber surface, or carried within a hollow fiber.

Fiber diameters can be of any suitable length in accordance withcharacteristics of the target or predetermined tissue in or at which theimplant is to be placed. Representative size ranges include a diameterof about 1 micron, about 5 microns, about 10 microns about 20 microns,about 40 microns, about 60 microns, about 80 microns, about 100 microns,about 120 microns, about 140 microns, about 160 microns, about 180microns, about 200 microns, about 220 microns, about 240 microns, about260 microns, about 280 microns, about 300 microns, about 320 microns,about 340 microns, about 360 microns, about 380 microns, about 400microns, about 450 microns or about 500 microns (including intermediatelengths). In various embodiments, the diameter of the fibers can be lessthan about 1 micron or greater than about 500 microns. Additionally,nanofibers fibers with diameters in the nanometer range (1-1000nanometers) are envisioned for certain embodiments. Additionally, largefibers with diameters up to 3.5 cm are envisioned for certainembodiments.

In some embodiments, representative fiber size ranges include 25 μm to100 μm in diameter. As would be apparent to one in ordinary skill in theart upon review of the present disclosure, 25 μm comprises approximatelythe size of a microsurgery suture. In some embodiments the diameter ofthe fibers provides just enough integrity for the fiber to be held undertension and therefore implemented in a process of making as disclosedherein.

In some embodiments, the distance between the fibers can range fromabout 1 micron to about 1,000 microns. For example, the distance betweenthe fibers can be about 5 microns, about 10 microns, about 50 microns,about 70 microns, about 90 microns, about 100 microns, about 120microns, about 140 microns, about 160 microns, about 180 microns, about200 microns, about 220 microns, about 240 microns, about 260 microns,about 280 microns, about 300 microns, about 320 microns, about 340microns, about 360 microns, about 380 microns, about 400 microns, about450 microns or about 500 microns. In various embodiments the distancebetween the fibers can be less than 1 micron or greater than 500microns.

In other embodiments of the presently disclosed subject matter, thefibers or subset of fibers, can contain one or more therapeutic agentssuch that the concentration of the therapeutic agent or agents variesalong the longitudinal axis of the fibers or subset of fibers. Theconcentration of the active agent or agents can vary linearly,exponentially or in any desired fashion, as a function of distance alongthe longitudinal axis of a fiber. The variation can be monodirectional;that is, the content of one or more therapeutic agents can decrease fromthe first end of the fibers or subset of the fibers to the second end ofthe fibers or subset of the fibers. The content can also vary in abidirectional fashion; that is, the content of the therapeutic agent oragents can increase from the first ends of the fibers or subset of thefibers to a maximum and then decrease towards the second ends of thefibers or subset of the fibers.

Thus, in some embodiments, the fibers serve as a degradable ornondegradable carrier to deliver one or more specific sequences ofgrowth factors, antibiotics, cytokines, etc. that are embedded withinthe fiber matter, attached to the fiber surface, or carried within ahollow fiber.

For fibers that contain one or more therapeutic agents, the agent oragents can include: a growth factor, an immunodulator, a compound thatpromotes angiogenesis, a compound that inhibits angiogenesis, ananti-inflammatory compound, an antibiotic, a cytokine, ananti-coagulation agent, a procoagulation agent, a chemotactic agent,agents that promotes apoptosis, an agent that inhibits apoptosis, amitogenic agent, a radioactive agent, a contrast agent for imagingstudies, a viral vector, a polynucleotide, therapeutic genes, DNA, RNA,a polypeptide, a glycosaminoglycan, a carbohydrate, a glycoprotein, andcombinations thereof.

In some embodiments, the three-dimensional fiber scaffold comprises a3-D textile scaffold. In this case, the fiber systems are referred to asyarn systems.

Fiber scaffolds suitable for inclusion with the presently disclosedsubject matter can be derived from any suitable source (e.g., matrigel),or any of a variety of commercial sources for suitable fiber scaffolds(e.g., polyglycolic acid can be obtained from sources such as Ethicon,Somerville, N.J., United States of America).

In some embodiments, the fiber scaffold can be prepared in a hydratedform or it can be dried or lyophilized into a substantially anhydrousform or a powder. Thereafter, the powder can be rehydrated for use as acell culture substrate, for example by suspending it in a suitable cellculture medium. In this regard, the fiber scaffold can be mixed withother suitable scaffold materials, such as described above.

In some embodiments, the fiber scaffold is biodegradable over time, suchthat it will be absorbed into the subject as it develops. Suitable fiberscaffolds, thus, can be formed from monomers such as glycolic acid,lactic acid, propyl fumarate, caprolactone, hyaluronan, hyaluronic acid,and the like. Other fiber scaffolds can include proteins,polysaccharides, polyhydroxy acids, polyorthoesthers, polyanhydrides,polyphosazenes, or synthetic polymers (particularly biodegradablepolymers). In some embodiments, suitable polymers for forming the fiberscaffold can include more than one monomer (e.g., combinations of theindicated monomers). Further, the fiber scaffold can include hormones,such as growth factors, cytokines, and morphogens (e.g., retinoic acid,arachidonic acid, etc.), desired extracellular matrix molecules (e.g.,fibronectin, laminin, collagen, etc.), or other materials (e.g., DNA,viruses, other cell types, etc.) as desired.

Polymers for use in the presently disclosed subject matter includesingle polymer, co-polymer or a blend of polymers of poly(L-lacticacid), poly(DL-lactic acid), polycaprolactone, poly(glycolic acid) orpolyanhydride. Naturally occurring polymers can also be used such asreconstituted or natural collagens or silks. Those of skill in the artwill understand that these polymers are just examples of a class ofbiodegradable polymer matrices that can be used in the presentlydisclosed subject matter. Further biodegradable matrices includepolyanhydrides, polyorthoesters, and poly(amino acids). Any such matrixcan be utilized to fabricate a biodegradable polymer matrix withcontrolled properties for use in the presently disclosed subject matter.

Exemplary natural polymers include naturally occurring polysaccharides,such as, for example, arabinans, fructans, fucans, galactans,galacturonans, glucans, mannans, xylans (such as, for example, inulin),levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins,including amylose, pullulan, glycogen, amylopectin, cellulose, dextran,dextrin, dextrose, glucose, polyglucose, polydextrose, pustulan, chitin,agarose, keratin, chondroitin, dermatan, hyaluronic acid, alginic acid,xanthan gum, starch and various other natural homopolymer orheteropolymers, such as those containing one or more of the followingaldoses, ketoses, acids or amines: erythrose, threose, ribose,arabinose, xylose, lyxose, allose, altrose, glucose, dextrose, mannose,gulose, idose, galactose, talose, erythrulose, ribulose, xylulose,psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose,sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine,cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid,lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaricacid, galacturonic acid, mannuronic acid, glucosamine, galactosamine,and neuraminic acid, and naturally occurring derivatives thereof.Accordingly, suitable polymers can include, for example, proteins, suchas albumin.

Exemplary semi-synthetic polymers include carboxymethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose,and methoxycellulose. Exemplary synthetic polymers includepolyphosphazenes, polyethylenes (such as, for example, polyethyleneglycol (including the class of compounds referred to as PLURONICS®,commercially available from BASF, Parsippany, N.-J., U.S.A.),polyoxyethylene, and polyethylene terephthlate), polypropylenes (suchas, for example, polypropylene glycol), polyurethanes, polyvinyl alcohol(PVA), polyvinyl chloride and polyvinylpyrrolidone, polyamides includingnylon, polystyrene, polylactic acids, fluorinated hydrocarbon polymers,fluorinated carbon polymers (such as, for example,polytetrafluoroethylene), acrylate, methacrylate, andpolymethylmethacrylate, and derivatives thereof.

The polymeric materials can be selected from those materials which canbe polymerized or their viscosity altered by application of exogenousmeans. For example, by application of light, ultrasound, radiation, orchelation, alone or in the presence of added catalyst, or by endogenousmeans, for example, a change to physiological pH, diffusion of calciumions (alginate) or borate ions (polyvinyl alcohol) into the polymer, orchange in temperature.

It is also to be understood that while in some embodiments, the one ormore cells of the presently disclosed subject matter can be employed inconjunction with the fibers of the 3-D fiber scaffold, it is alsoenvisioned that the one or more cells can be present in a matrixcomprising a gel or polymer phase without a fiber scaffold. Further, itis to be understood that in some embodiments, thedifferentiation-promoting factor (e.g., BMP-6) can be incorporated intothe 3-D fiber scaffold for controlled release over time.

III.B. Three-Dimensional Weaving

In some embodiments, the presently disclosed subject matter provides anovel 3-D weaving technology that can be employed to form compositeanatomically-shaped biomaterial scaffolds that can be impregnated with agelatinous material (fibrin, gelatin, alginate, agarose, etc.) topromote cell growth. The fiber-reinforced scaffold can be woven toreproduce the biomechanical properties of both cartilage and bone andcan be coated with biologically active factors in a site-specific mannerto promote cell differentiation, growth, and activity as required. Onerepresentative form of the scaffold is a largely hemispherical two-phase(bone/cartilage) construct. Other embodiments can include a “saddle”shaped two-phase (bone/cartilage) construct. Other shapes can be made tomimic more complex joint architecture including, but not limited to, theknee joint, with multiple tissues (bone/cartilage/meniscus).

An advantage of presently disclosed weaving technology is that thelarge, ordered, and interconnected pores or interstices of the 3-D weaveallow for consistent and even distribution of cells (including but notlimited to ADS or ADS-derived cells) throughout the composite scaffold.The interstices comprise a pore size ranging in some embodiments about 1μm to about 1,000 μm, in some embodiments about 5 μm to about 750 μm, insome embodiments from about 10 μm to about 500 μm, in some embodimentsfrom about 25 μm to about 250 μm, and in some embodiments from about 50μm to about 125 μm. Such a structure provides sufficient area onto whichthe cells can grow and proliferate.

Three-dimensional fiber scaffolds are produced in some embodiments usinga 3-D weaving loom, specifically constructed to produce precisestructures from fine diameter fibers. A computer controlled weavingmachine can produce true 3-D shapes by placing fibers axially (x-warpdirection), transversely (y-weft, or filling direction), and vertically(z-thickness direction). Multiple layers of warp yarns are separatedfrom each other at distances that allow the insertion of the weft layersbetween them. Two layers of Z-yarns, which are normally arranged in thewarp direction, are moved (after the weft insertion) up and down, indirections opposite to the other. This action is followed by the“beat-up”, or packing of the weft into the scaffold, and locks the twoplanar fibers (the warp and weft) together into a uniform configuration.Change of yarn densities can be achieved for warp by altering the reeddensity and warp arrangement and for weft by varying the computerprogram controlling the take-up speed of a stepper motor.

In some embodiments, the three-dimensional fiber scaffold comprisesthree orthogonally woven fiber systems, a plurality of braided fibersystems, a plurality of circular woven fiber systems, or combinationsthereof.

In some embodiments the presently disclosed subject matter comprises a3-D weave of fibers in three orthogonal directions. In comparison tostandard weaving methods, this process eliminates fiber crimp and formsa true 3-D structure. In general, most current 3-D textile compositesare constructed by laminating multiple 2-D structures together, and thelamination interface between multiple layers is the weak point in thecomposite where debonding or delamination can always occur. Becausethere is no “crimping” of the in-plane fibers as in a standard wovenmatrix, the straightness of the presently disclosed scaffolds decreasesbuckling of individual fibers and significantly improves their strengthand stiffness properties under both compressive and tensile stresses.

An advantage of the presently disclosed weaving technique is that eachfiber can be selected individually and woven into a construct. Usingthis method of assembly, customized structures can be easily created byselectively placing different constituent fibers (e.g., fibers ofvarious material composition, size, and/or coating/treatment) throughoutthe scaffold. In this manner, physical and mechanical properties of thescaffold can be controlled (i.e., pore sizes can be selected,directional properties can be varied, and discreet layers can beformed). Using this technique, the inhomogeneity and anisotropy ofvarious tissues can be reproduced by constructing a scaffold that mimicsthe normal stratified tissue network using a single, integral scaffold.

Setting of the yarn systems can be done via any of a number ofart-recognized techniques, including but not limited to ultrasonication,a resin, infrared irradiation, heat, or any combination thereof. Settingof the yarn systems within the scaffold in this manner providescuttability and suturability. Sterilization can be performed by routinemethods including, but not limited to autoclaving, radiation treatment,hydrogen peroxide treatment, ethylene oxide treatment, and the like.

Representative methods for making three-dimensional textile structuresare also disclosed in U.S. Pat. Nos. 5,465,760 and 5,085,252, thecontents of each of which are incorporated herein by reference in theirentireties. The following patent publications are also incorporatedherein by reference in their entireties: PCT International PatentApplication Publication WO 01/38662 (published May 31, 2001); PCTInternational Patent Application Publication WO 02/07961 (published Jan.31, 2002); U.S. Patent Application Publication 2003/0003135 (publishedJan. 2, 2003), and PCT International Patent Application Serial No.PCT/US06/14437, filed Apr. 18, 2006.

III.C. Consolidation of Fiber Scaffolds with Cell-Seeded Hydrogel

As discussed herein above, the presently disclosed subject matterprovides in some embodiments a 3-D woven fiber scaffolds for use injoint replacement. The scaffold can be used in its native form, as acomposite material in combination with other materials, as an acellular(non-viable) matrix, or combined with cells (such as but not limited toADS and/or ADS-derived cells) and/or growth modulating materials (e.g.,growth factors) for use in repair, regeneration, and/or replacement ofdiseased or traumatized tissue (e.g., a joint) and/or tissue engineeringapplications. An advantage of the presently disclosed subject matter isthe ability to produce biomaterial scaffolds and composite matrices thathave precisely defined mechanical properties that can be inhomogeneous(vary with site), anisotropic (vary with direction), nonlinear (varywith strain), and/or viscoelastic (vary with time or rate of loading).By combining a fiber-based scaffold with a biocompatible resin ormatrix, another advantage of the composite matrix is that themicroenvironment of embedded cells can be controlled to promoteappropriate cell growth and/or activity while providing for theprescribed mechanical properties. These characteristics can arise fromthe combination of the two components.

In some embodiments, the fiber scaffold is mixed with cells (such as butnot limited to ADS or ADS-derived cells) and crosslinked to form ahydrogel matrix containing the cells before or after implantation intothe body. The scaffold functions to provide a template for theintegrated growth and differentiation of the desired tissue. In someembodiments, a polymer forms the hydrogel within the body upon contactwith a crosslinking agent. A hydrogel is defined as a substance formedwhen an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic or hydrogen bonds to create a three-dimensionalopen-fiber scaffold structure which entraps water molecules to form agel. Naturally occurring and synthetic hydrogel forming polymers,polymer mixtures and copolymers can be utilized as hydrogel precursors.See, for example, U.S. Pat. No. 5,709,854 and WO 94/25080.

Hydrogels can be classified into two broad categories: reversible orphysical and irreversible or chemical. The networks in physical gels areheld together by molecular entanglements and/or secondary forcesincluding ionic, hydrogen bonding or hydrophobic forces. Physicalhydrogels are characterized by significant changes in the rheologicalproperties as a function of temperature, ionic concentration, anddilution. Chemical gels, also called permanent gels, are characterizedby chemically crosslinked networks. When crosslinked, these gels reachan equilibrium swelling level in aqueous solutions which depends mainlyon the crosslink density.

The preparation of hydrogels can be achieved by a variety of methodswell known to those of ordinary skill in the art. Physical gels can beformed by: heating or cooling certain polymer solutions (cool agarose,for example), using freeze-thaw cycles to form polymer microcrystals,reducing the solution pH to form a hydrogen-bonded gel between twodifferent polymers in the same aqueous solution, mixing solutions of apolyanion and a polycation to form a complex coacervate gel, gelling apolyelectrolyte solution with a multivalent ion of opposite charge,reticulation of linear polymers, grafting of synthetic polymers ontonaturally occurring macromolecules, and chelation of polycations(Hoffman (2000) Advanced Drug Delivery Reviews, 43:3-12). Chemical gelscan be created by crosslinking polymers in the solid state or insolution with radiation, chemical crosslinkers like glutaraldehyde, ormultifunctional reactive compounds. They can also be made bycopolymerizing a monomer and a crosslinker in solution, copolymerizing amonomer and a multifunctional macromer, polymerizing a monomer within adifferent solid polymer to form an IPN gel, or chemically converting ahydrophobic polymer to a hydrogel (Hennick and van Nostrum (2002)Advanced Drug Delivery Reviews, 54:13-26).

The presently disclosed subject matter, in some embodiments, providesthe use of hydrogel precursor materials and non-gelling proteins andpolysaccharides as scaffold materials themselves or within the core ofthe fibers. Hydrogel precursor materials are the same materials as thosethat form hydrogels, but they are not exposed to the agents orconditions that normally gel the materials, or can be other proteins andpolysaccharides that form gels but not hydrogels. For example, alginatesalts, such as sodium alginate, are gelled in the presence of divalentcations, such as calcium, while other materials create hydrogels via achange in pH or temperature. Certain embodiments of the presentlydisclosed subject matter comprise the use of precursor materials thatare never gelled. Other embodiments of the presently disclosed subjectmatter comprise the use of precursor materials in the fabricationprocess that later can form gels or hydrogels. The formation of gels orhydrogels in the fiber layer can take place as a part of the fiberfabrication process, after the fiber has been fabricated, or after theapplication of an appropriate type of external stimuli, includingplacing the fiber in vitro or in vivo. The terms “gel” or “hydrogel” asused herein is intended to include the formed gel or hydrogel as well asthe appropriate precursor molecules involved in the formation of gelsand hydrogels.

An exemplary method for combining the fiber-based scaffolds with a gelmatrix is via the utilization of a vacuum-assisted molding process.Particularly, the technique utilizes vacuum pressure to draw the gelwhile still in its liquid form into the 3-D fiber scaffold, effectivelyfilling the pore spaces and encapsulating the fibers. Once the gel hascompletely infused the scaffold, it is solidified by an appropriatecross-linking method to form the composite construct. In someembodiments, cells and/or growth promoting materials are seeded into thescaffolds by mixing them into a liquid gel prior to infusion into ascaffold.

Thus, the 3-D fiber performs, which in some embodiments are 3-Dorthogonally woven fiber performs, can be infiltrated with a cell-seededor acellular gel material to form a composite construct or bioartificialimplant. In some embodiments, the cells can be primary cells (e.g.,chondrocytes, osteoblasts, fibroblasts, etc.) and/or undifferentiatedprogenitor cells (e.g., stem cells, including but not limited to ADScells). The gel biomaterial can be one of many different types ofcrosslinkable, photocrosslinkable, temperature sensitive, and/or othergel that can sustain cell growth and provide mechanical function to thescaffold. Possible gels include fibrin, alginate, agarose, elastin,chitosan, collagen, etc.

In some embodiments, to form the fiber scaffold, the cells areintroduced onto the scaffold such that they permeate into theinterstitial spaces therein. For example, the matrix can be soaked in asolution or suspension containing the cells, or they can be infused orinjected into the matrix. In some embodiments, a hydrogel is formed bycrosslinking a suspension comprising the fiber and the inventive cellsdispersed therein. This particular method of formation permits the cellsto be dispersed throughout the fiber scaffold, facilitating more evenpermeation of the fiber scaffold with the cells. As would be readilyapparent to one of ordinary skill in the art, the composition caninclude mature cells of a desired phenotype or precursors thereof,particularly to potentate the induction of the inventive stem cells todifferential appropriately within the fiber scaffold (e.g., as an effectof co-culturing such cells within the fiber scaffold).

In some embodiments, cells can be employed to seed the scaffold, whichprovides a template for the integrated growth and differentiation intotissue capable of substantially functioning as cartilage and bone. Byforming an integrated “bone-cartilage” construct in the shape of a joint(e.g., a hip) outside the body, the implant can adhere to the bonesurface of the joint and integrate appropriately. As would be readilyapparent to one of skill in the art, cell types, such as ADS orADS-derived cells, mesenchymal stem cells, primary chondrocytes, orosteoblasts are useful for these applications.

In some embodiments the scaffold can be coated on one or more surfaces,before or after consolidation with a gel and/or cells, with a materialto improve the mechanical, tribological, or biological properties of thecomposite. Such a coating material can be resorbable or non-resorbableand can be applied by dip-coating, spray-coating, electrospinning,plasma spray coating, and/or other coating techniques. The material canbe a single or multiple layers or films. The material can also compriserandomly aligned or ordered arrays of fibers. In some embodiments, thecoating can comprise electrospun nanofibers. The coating material can beselected from the group including, but not limited to polypropylene,polyester, polytetrafluoroethylene (PTFE), expanded PTFE (ePTFE),polyethylene, polyurethane, polyamide, nylon, polyetheretherketone(PEEK), polysulfone, a cellulosic, fiberglass, an acrylic, tantalum,polyvinyl alcohol, carbon, ceramic, a metal, polyglycolic acid (PGA),polylactic acid (PLA), polyglycolide-lactide, polycaprolactone,poly(ethylene glycol) (PEG), polydioxanone, polyoxalate, apolyanhydride, a poly(phosphoester), catgut suture, collagen, silk,chitin, chitosan, hydroxyapatite, bioabsorbable calcium phosphate,hyaluronic acid, elastin, lubricin, and combinations thereof.

In some embodiments a smooth surface coat on the scaffold is thusprovided if needed. In some embodiments, the surface coat can increasedurability and/or reduce friction of and/or at the surface.

In some embodiments, a fiber scaffold can be employed in any suitablemanner to facilitate the growth and generation of the desired tissuetypes or structures. For example, the scaffold can be constructed usingthree-dimensional or stereotactic modeling techniques. Thus, forexample, a layer or domain within the scaffold can be populated by cellsprimer for one type of cellular differentiation, and another layer ordomain within the scaffold can be populated with cells primed for adifferent type of cellular differentiation. As disclosed herein and aswould be readily apparent to one of skill in the art, to direct thegrowth and differentiation of the desired structure, in someembodiments, the scaffold can be cultured ex vivo in a bioreactor orincubator, as appropriate. In some embodiments, the structure isimplanted within the subject directly at the site in which it is desiredto grow the tissue or structure. In further embodiments, the scaffoldcan be grafted on a host (e.g., an animal such as a pig, baboon, etc.),where it can be grown and matured until ready for use, wherein themature structure is excised from the host and implanted into thesubject.

Thus, provided in some embodiments is a novel scaffold for the growth oftissues/organs both in vitro and in vivo. In particular embodiments,provided is a biodegradable scaffold of multiple layers made preferablywith collagen or collagen composite material to be placed in either abioreactor or a directly into a living bio-organism for the purpose ofreplacing a damaged and/or missing organ such as bone, wherein thescaffold comprises mechanical structures for stimulating cells.

In some embodiments, the presently disclosed subject matter providesmethods for producing an implant for use in joint resurfacing. In someembodiments, the method comprises forming a three-dimensional fiberscaffold, the scaffold comprising at least three systems of fibers;wherein (i) two of the three fiber systems define an upper layer, alower layer and a medial layer between the upper layer and the lowerlayer within the three-dimensional fiber scaffold; (ii) one of the atleast three fiber systems interconnects the upper layer, the lower layerand the medial layer; and (iii) the at least three fiber systems eachcomprise a biocompatible material. One or more cells can be disposed inthe fiber scaffold such that the cells/matrix construct can develop intotissue capable of substantially functioning as bone, cartilage, or boneand cartilage. It is to be understood that the fiber scaffold or one ormore of the fiber systems can provide one or more characteristics ofjoint to be replaced upon implantation.

In some embodiments, the scaffold, before or after seeding with cells,is molded into the appropriate shape using any standard manufacturingmethods including, but not limited to block molding, shape molding,vacuum molding, press molding, compression molding, and combinationsthereof.

In some embodiments, a portion or all of the cells seeded in thescaffold are killed (i.e. devitalized) and/or removed prior toimplantation. The scaffold can also be treated with DNase, RNase, and/orother enzymes to degrade and/or remove any nucleic acids or geneticmaterial before implantation. Thus, in some embodiments an artificial“tissue” derived from the cell-seed scaffold is provided.

The presently disclosed subject matter also provides methods forreplacing a predetermined joint in a subject. In some embodiments, themethod comprises (a) providing a joint replacement implant comprising:(i) a three-dimensional fiber scaffold formed of at least three systemsof fibers, wherein (1) two of the three fiber systems define an upperlayer, a lower layer and a medial layer between the upper layer and thelower layer within the three-dimensional fiber scaffold; (2) one of theat least three fiber systems interconnects the upper layer, the lowerlayer and the medial layer; and (3) the at least three fiber systemseach comprise a bio-compatible material; and (ii) one or more cells thatcan develop into tissue capable of substantially functioning as bone,cartilage, or bone and cartilage, wherein the fiber scaffold, or one ormore of the fiber systems, provide one or more characteristics of thepredetermined joint upon implantation; and (b) implanting at a site ofthe predetermined joint in the subject the implant provided in step (a)to thereby replace a joint in the subject. In some embodiments, thepredetermined joint is selected from the group consisting of a hipjoint, a knee joint, a shoulder joint, an ankle joint, and an elbowjoint, although the methods and compositions disclosed herein are notrestricted to just these joints.

IV. Bioreactor for Tissue Growth and Differentiation

In some embodiments, a bioreactor is used to enhance growth anddifferentiation of the cells. The bioreactor can enhance tissuedifferentiation by controlling the temperature, carbon dioxide, oxygen,and nitrogen concentrations, physicochemical environment (e.g., pH,oxygen tension, osmolarity), perfusion, and mechanical loadingenvironment. The bioreactor simultaneously can provide dynamiccompressive loading to the joint replacement implants as they grow invitro.

Thus, as is known to those skilled in the art, bioreactors help inestablishing spatially uniform cell distribution on three-dimensionalscaffolds, maintaining desired concentrations of gases and nutrients inthe culture medium, providing sufficient mass transfer to growingtissues, and exposing developing tissues to physical stimuli.

For example, one or more ADS and/or ADS-derived cells can be grownand/or differentiated in the bioreactor in any suitable cell culturemedium. Typically, cell culture media comprise a base medium such asDulbecco's Modified Eagle's Medium (DMEM) and/or Ham's Nutrient MixtureF12 (F12) medium supplemented with one or more additives selected fromthe group consisting of an animal serum (e.g., bovine serum) or areduced serum supplement (e.g., OPTI-MEM® I reduced serum mediumsupplement, Invitrogen Corp., Carlsbad, Calif., United States ofAmerica), an antibiotic (e.g., penicillin and/or streptomycin), and oneor more amino acids such as glutamine. Other additives that can beemployed are known to those skilled in the art, and can includeinsulin/transferrin/selenium supplement (ITS, available from InvitrogenCorp., Carlsbad, Calif., United States of America), essential andnon-essential amino acids, salts, buffers, and peptides and polypeptidessuch as growth factors, cytokines, etc. Upon a review of the presentdisclosure, the skilled artisan will understand how to optimize theconcentrations of the various components in order to facilitate thegrowth and/or differentiation of the cells.

In some embodiments, the cell culture medium further comprises a growthmodulating material. As used herein, the phrase “growth modulatingmaterial” refers to a molecule or group of molecules that individuallyor in combination promotes the growth, survival, and/or differentiationof the one or more cells that can develop into a tissue of apredetermined site, such as but not limited to bone, cartilage, or bothbone and cartilage. Typically, although not exclusively, the growthpromoting material can be present in the medium and/or on or in thescaffold on which the one or more cells is growing.

In some embodiments, the implantable composition can be maintained inthe bioreactor prior to implantation for a time sufficient to providetissue comprising tissue capable of replacing tissue at a predeterminedsite, for example but not limited to bone, cartilage, or both bone andcartilage.

In some embodiments, the bioreactor provides an in vitro environmentthat embodies chemical and mechanical signals that regulate tissuedevelopment and maintenance in viva. The bioreactor culture vessels caninclude, but are not limited to, spinner flasks, rotating vessels, aperfused chamber, or a perfused column. The bioreactor thus can have theability to apply a variety of (mechanical) signals to the cells.

Bioreactors, especially bioreactors used for tissue regenerationprocesses, are well known. Reference is hereby made, e.g., to U.S. Pat.Nos. 6,306,169, 6,197,575, 6,080,581, 5,677,355, 5,433,909, 5,898,040,and the like, which are hereby incorporated by reference.

V. Formulation

The compositions of the presently disclosed subject matter comprise insome embodiments a composition that includes a carrier, particularly apharmaceutically acceptable carrier. As disclosed herein above, anysuitable pharmaceutical formulation can be used to prepare thecompositions for administration to a subject.

For example, suitable formulations can include aqueous and non-aqueoussterile injection solutions that can contain anti-oxidants, buffers,bacteriostatics, bactericidal antibiotics and solutes which render theformulation isotonic with the bodily fluids of the intended recipient;and aqueous and non-aqueous sterile suspensions which can includesuspending agents and thickening agents. The formulations can bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and can be stored in a frozen or freeze-dried(lyophilized) condition requiring only the addition of sterile liquidcarrier, for example water for injections, immediately prior to use.Some exemplary ingredients are SDS, in one example in the range of 0.1to 10 mg/ml, in another example about 2.0 mg/ml; and/or mannitol oranother sugar, for example in the range of about 10 to 100 mg/ml, inanother example about 30 mg/ml; and/or phosphate-buffered saline (PBS).

It should be understood that in addition to the ingredients particularlymentioned herein, the formulations of the presently disclosed subjectmatter can include other agents conventional in the art with regard tothe type of formulation in question. For example, sterile pyrogen-freeaqueous and non-aqueous solutions can be used.

The therapeutic regimens and compositions of the presently disclosedsubject matter can also be used with additional adjuvants or biologicalresponse modifiers including, but not limited to, cytokines and otherimmunomodulating compounds.

VI. Administration

Administration of the compositions of the presently disclosed subjectmatter can be by any method known to one of ordinary skill in the art.In some embodiments, suitable methods for administration of the cells ofthe presently disclosed subject matter include, but are not limited toinjection into the target tissue or target site. The term “targettissue” as used herein refers to an intended site for engraftmentfollowing administration to a subject.

In some embodiments, the compositions comprise cells present in a matrix(e.g., a gel) within the pores of a fiber scaffold. The fiber scaffoldcan be implanted at a pre-determined site (i.e., a joint) to replace,repair, and/or restore a target tissue and/or structure at theparticular site of insertion. In some embodiments, the fiber scaffoldcan be implanted in a subject to alleviate tissue loss, damage, injury,or combinations thereof.

The fiber scaffolds can be implanted into the subject at the site inneed of treatment using standard surgical techniques. In someembodiments, the fiber scaffold is constructed, seeded with cells andcultured in vitro prior to implantation. The cells can be cultured inthe device, tested for viability, and then implanted. In someembodiments, the fiber scaffold is constructed, seeded with cells andcultured in vivo after or during implantation. In some embodiments, thescaffold is implanted without cells.

In some embodiments, the fiber scaffolds can be used for delivery ofmultiple different cell types. The scaffold can be implanted in one ormore different areas of the body to suit a desired application.

In addition, there are situations where it could be desirable to usemore than one matrix, each implanted at the most optimum time for growthof the attached cells to form a functioning three-dimensional structurefrom the different matrices.

VII. Dose

An effective dose of a composition of the presently disclosed subjectmatter is administered to a subject in need thereof. A “treatmenteffective amount” or a “therapeutic amount” is an amount of atherapeutic composition (e.g., induced ADS cells in a pharmaceuticallyacceptable carrier or excipient) sufficient to produce a biologically orclinically relevant response in a subject being treated. The actualnumber of induced ADS cells, as an example, in the compositions of thepresently disclosed subject matter can be varied so as to administer anumber of the induced ADS cells that is effective to achieve the desiredtherapeutic response for a particular subject. The selected dosage levelwill depend upon several factors including, but not limited to theability of the induced ADS cells or their progeny to engraft the targettissue, the route of administration, combination with other drugs ortreatments, the severity of the condition being treated, and thecondition and prior medical history of the subject being treated.

The potency of a composition can vary, and therefore a “treatmenteffective amount” can vary. However, using standard assay methods, oneskilled in the art can readily assess the potency and efficacy of theinduced ADS cells of the presently disclosed subject matter, and adjustthe therapeutic regimen accordingly. After review of the disclosure ofthe presently disclosed subject matter presented herein, one of ordinaryskill in the art can tailor the dosages to an individual subject, takinginto account the particular formulation, method of administration to beused with the composition, and particular disease treated. Furthercalculations of dose can consider subject height and weight, severityand stage of symptoms, and the presence of additional deleteriousphysical conditions. Such adjustments or variations, as well asevaluation of when and how to make such adjustments or variations, arewell known to those of ordinary skill in the art of medicine.

VIII. Subjects

The subjects treated in the presently disclosed subject matter are insome embodiments human subjects, although it is to be understood thatthe presently disclosed subject matter is effective with respect to allvertebrate animals, including mammals, which are intended to be includedin the term “subject”. Moreover, a mammal is understood to include anymammalian species in which treatment or prevention of a disease isdesirable, particularly agricultural and domestic mammalian species.

More particularly provided is the treatment of mammals such as humans,as well as those mammals of importance due to being endangered (such asSiberian tigers), of economic importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including the treatmentof those kinds of birds that are endangered, kept in zoos, as well asfowl, and more particularly domesticated fowl, for example, poultry,such as turkeys, chickens, ducks, geese, guinea fowl, and the like, asthey are also of economic importance to humans. Thus, contemplated isthe treatment of livestock, including, but not limited to, domesticatedswine (pigs and hogs), ruminants, horses, poultry, and the like.

IX. Kits

All the essential materials and reagents required for the variousaspects of the presently disclosed subject matter can be assembledtogether in a kit. The kits typically include vials comprising thedesired components in close confinement for commercial sale such as in,e.g., injection or blow-molded plastic containers. Irrespective of thenumber or type of containers, the kits of the presently disclosedsubject matter can be typically packaged with instructions for use ofthe kit components.

As discussed above, the cells, populations, scaffolds, and compositionsof the presently disclosed subject matter can be used in tissueengineering and regeneration. The disclosed scaffolds can convenientlybe employed as part of a cell culture kit. Accordingly, the presentlydisclosed subject matter can provide a kit including the presentlydisclosed scaffolds and one or more other components, such as hydratingagents (e.g., water, physiologically-compatible saline solutions,prepared cell culture media, serum or derivatives thereof etc.), cellculture substrates (e.g., culture dishes, plates, vials, etc.), cellculture media (whether in liquid or powdered form), antibioticcompounds, hormones, and the like. While the kit can include any suchingredients, it can include all ingredients necessary to support theculture and growth of desired cell types upon proper combination. Ofcourse, if desired, the kit also can include cells (typically frozen),which can be seeded into the fiber scaffold as described herein.

By way of example, any of the steps for isolating one of the cellsources disclosed in the presently disclosed subject matter can alsoprovide a kit for isolating such reagents from adipose tissues. The kitcan include a device for isolating adipose tissue from a patient (e.g.,a cannula, a needle, an aspirator, etc.), as well as a device forseparating stem cells (e.g., through methods described herein or throughmethods commonly known by one of ordinary skill in the art). The kit canbe employed, for example, as a bedside source of stem cells that canthen be re-introduced from the same individual as appropriate. Thus, thekit can facilitate the isolation of ADS cells for implantation in apatient needing regrowth of a desired tissue type, even in the sameprocedure. In this respect, the kit can also include a medium fordifferentiating the cells, such as those set forth herein. Asappropriate, the cells can be exposed to the medium to prime them fordifferentiation within the patient as needed. In addition, the kit canbe used as a convenient source of stem cells for in vitro manipulation(e.g., cloning or differentiating as described herein). In someembodiments, the kit can be employed for isolating a fiber scaffold asdescribed herein.

EXAMPLES

The following Examples have been included to illustrate modes of thepresently disclosed subject matter. In light of the present disclosureand the general level of skill in the art, those of skill willappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Materials and Methods using in the Examples

Cell Culture. hADS cells from three female donors were purchased fromZen-Bio Inc. (Durham, N.C., United States of America). All hADS cellswere originally obtained from subcutaneous abdominal adipose tissue, andall donors were non-smokers and non-diabetics. Lot L012202 was derivedfrom a 34 year old donor with BMI of 22.24; L062801 from a 37 year oldwith BMI of 23.29; and L031502 from a 47 year old with BMI of 29.08. Thecells were plated on 225 cm² culture flasks (Corning, Corning, N.Y.,United States of America) at an initial density of 8,000 cells/cm² inexpansion medium.

Expansion medium comprised DMEM/F12 (Cambrex Bio Science, Walkersville,Md., United States of America), 10% FBS (Hyclone, Logan Utah, UnitedStates of America), 1% penicillin-streptomycin-fungizone (InvitrogenGIBCO® Corp., Carlsbad, Calif., United States of America), 0.25 ng/mlTGF-β1 (R&D Systems, Minneapolis, Minn., United States of America), 5ng/ml EGF (Roche Diagnostics, Indianapolis, Ind., United States ofAmerica), and 1 ng/ml bFGF (Roche Diagnostics, Indianapolis, Ind.,United States of America).

Culture media was replaced every other day, and the cultures wereallowed to reach 90% confluence before trypsinizing and replating at8000 cell/cm². The hADS cells were passaged to cell stage P4 at whichpoint they were trypsinized off the culture plates and resuspended in1.2% alginate solution at 5×10⁶ cells/mL. Using a 1-mL pipetter, thealginate-cell suspension was dropped into a 102 mM CaCl₂ solution makingspherical alginate beads. Each bead was approximately 0.4 cm indiameter, containing approximately 150,000 cells.

The hADS cells were then cultured in seven different culture conditionsfor seven days. One of the culture conditions, which served as acontrol, consisted of DMEM-high glucose (Invitrogen GIBCO® Corp.,Carlsbad, Calif., United States of America), 10% FBS, 1%penicillin-streptomycin, and ascorbic-2-phosphate (37.5 μg/ml). Fordifferentiation induction, 1% ITS+premix (0.62 μg/ml insulin, 0.62 μg/mltransferrin, and 0.62 ng/ml selenium; Collaborative Biomedical, BectonDickinson, Bedford, Mass., United States of America) was added to thecontrol medium in addition to the growth factors listed in Table 1. Thealginate beads were cultured in 24-well tissue culture plates (CorningLife Sciences, Corning, N.Y., United States of America) with three beadsper well and with 1 mL of medium in each well. Culture medium wasreplaced every other day.

TABLE 1 Growth Factor Combinations for ADS Cell Differentiation GrowthFactors Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 100 nM X DEX 10ng/ml X X TGF-β1 10 ng/ml X X X TGF-β3 100 ng/ml X X IGF-1 500 ng/ml X XrhBMP-6

DNA, [³H]-proline, and [³⁵S]-sulfate Assays. For the last 24 hours ofculture, 10 μCi/mL of [³H]-proline and 5 μCi/mL of [³⁵S]-sulfate wereadded to each of the different culture conditions in order to quantifytotal protein and GAG synthesis, respectively. After the beads weredigested in papain, DNA content (per three beads) was quantified usingthe PICOGREEN® fluorescent dsDNA assay (Molecular Probes, Eugene, Oreg.,United States of America). Radiolabel incorporation was quantified usinga scintillation analyzer, and the resulting data were normalized to DNAcontent. ANOVA was used with Fisher's PLSD post-hoc test to determinestatistical significance between the different conditions (α=0.05).

RNA Isolation and Real Time PCR. Following seven days in culture, thehADS cells were released from alginate using a solution of 150 mM NaCland 55 mM Na Citrate. RNA from these cells as well as from hADS cellsfrozen down at Day 0 of the experiment was obtained using the RNeasy®Mini kit from Qiagen (Valencia, Calif., United States of America) andwas treated with an RNAse-free DNAse (Qiagen). cDNA was synthesized fromRNA using ISCRIPT™ reverse transcriptase PCR (Bio-Rad, Hercules, Calif.,United States of America).

Using commercially bought primer-probes from Applied Biosystems (FosterCity, Calif., United States of America), real time PCR was used tocompare the resulting cDNA for five different genes: 18S rRNA(endogenous control), aggrecan, type I collagen, type II collagen, andtype X collagen. The amount of cDNA added per real time PCR reaction wasstandardized to 40 ng cDNA. The 2^(−ΔΔCt) method was used for relativequantification of gene expression Livak and Schmittqen (2001) Methods25:402-408) to compare the effects of the seven different cultureconditions on hADS cell gene expression. ANOVA was used with Fisher'sPLSD post-hoc test to determine statistical significance between thedifferent conditions (α=0.05).

Immunohistochemistry. After seven days in culture, alginate beads fromeach of the seven conditions and from each of the three donors werefixed for four hours in a solution of 4% paraformaldehyde, 100 mM sodiumcacodylate, and 50 mM BaCl₂ (the latter to irreversibly crosslink thealginate matrix) and then washed overnight in a 100 mM sodiumcacodylate, 50 mM BaCl₂ buffer. The beads were dehydrated with a seriesof increasing ethanol concentrations. The beads were then cleared withxylene and then embedded in paraffin wax.

Immunohistochemistry was performed on 5 μm sections using monoclonalantibodies to type I collagen (Sigma Chemical Co., St. Louis, Mo.,United States of America), type II collagen (II-II6B3 AB, DevelopmentalStudies Hybridoma Bank, The University of Iowa, Iowa City, Iowa, UnitedStates of America), type X collagen (Sigma), and chondroitin sulfate(3B3 antibody, gift from Dr. Virginia Kraus, Duke University MedicalCenter, Durham, N.C., United States of America). DIGEST-ALL™ (ZymedLaboratories, South San Francisco, Calif., United States of America) wasused for pepsin digestion on all sections except those stained forchondroitin sulfate. Sections to be stained for chondroitin sulfate weretreated with trypsin (Sigma), then with soybean trypsin inhibitor(Sigma), and then with chondroitinase (Sigma) to allow the antibody tointeract with a chondroitin-4-sulfate epitope.

HISTOSTAIN®-Plus ES Kit (Zymed) was used on all sections for blocking,secondary antibody staining, and subsequent linking to horseradishperoxidase. Aminoethyl carbazole (Zymed) was used as the enzymesubstrate/chromogen. The appropriate positive controls for each antibodywere prepared and examined to ensure antibody specificity: porcinecartilage for type II collagen and chondroitin sulfate, deep layer andcalcified zone of cartilage for type X collagen, and meniscus for type Icollagen. Negative controls showed minimal background staining.

Example 1 DNA Analysis

The growth factor and cytokine combinations disclosed herein resulted insignificant differences in hADS cells encapsulated within 1.2% alginate.In order to normalize the [³H]-proline and [³⁵S]-sulfate incorporationresults and also to evaluate the viability of the cells, dsDNA wasmeasured at the time of encapsulation in alginate and also at day 7, theterminal time point of the study (FIG. 1).

A two-factor ANOVA showed significant DNA differences between donors andgrowth factor conditions as well as an interactive effect between thedonor and growth factor combination (p<0.0001). A Fisher's PLSD post hoccomparison also demonstrated significant differences between allconditions except in the following conditions (p>0.05): control mediumand TGF-β3+IGF-I; TGF-β1 and TGF-β3; and TGF-β3+IGF-I+BMP-6 and BMP-6.

Example 2 Biosynthetic Activity

Significant differences in biosynthetic activity were also observed (seeFIGS. 2A and 2B). As with the DNA data, a two-factor ANOVA showedsignificant differences between donors and growth factor conditions aswell as an interactive effect between the donor and growth factorcombination (p<0.0001). Notably, [³H]-proline incorporation was greatestwithin the dexamethasone+TGF-β1 group, and this combination resulted ina statistically significant increase in [³H]-proline incorporationcompared to the TGF-β1 condition alone (p<0.0001).

The addition of BMP-6 to TGF-β3 and IGF-I also resulted in a significantincrease in protein biosynthetic activity compared to the other growthfactor combinations using TGF-β1 or TGF-β3 (without dexamethasone)(p<0.0001). All growth factor combinations resulted in significantincreases of [³⁵S]-sulfate incorporation compared to the control mediumalone (p<0.0001). Significant differences in [³⁵S]-sulfate incorporationwere also noted between the growth factor conditions containing BMP-6compared to all other conditions (p<0.05).

Example 3 Gene Expression

As measured by quantitative RT-PCR, the growth factor combinationsdisclosed herein displayed varying capabilities to inducedifferentiation with hADS cells encapsulated in 1.2% alginate. As hasbeen noted, mRNAs for two positive markers of chondrogenesis wereanalyzed (aggrecan and collagen II), and mRNAs of two negative markersof chondrogenesis were analyzed (collagen I and collagen X). The resultsare expressed as relative quantification of mRNA levels compared tocells at the time of encapsulation (Day 0). As an internal endogenouscontrol for each gene transcript, expression of 18s rRNA was alsomeasured. Again, the data are represented using the 2^(−ΔΔCt) method(Livak & Schmittgen, (2001) Methods 25:402-408), where the levels of 18Sare used to normalize the amount of mRNA transcript for each gene in thecontrols (Day 0 cells) and the experimental groups at Day 7. Theresulting data represent the fold increase or decrease in geneexpression for each gene transcript relative to Day 0 cells (FIGS. 3-6).For all of the genes studied (FIG. 3-6), two factor ANOVA analysesrevealed significant effects of both donor and growth factor conditionsas well as an interactive effect between donor and growth factorconditions (p<0.0001).

For aggrecan gene expression, only the BMP-6 condition resulted instatistically significant differences in gene expression versus allother conditions (p<0.0001). The addition of BMP-6 to the control mediumresulted in an average increase in aggrecan gene expression across the 3donors of approximately 200 fold. Interestingly, this same statisticallysignificance increase in aggrecan gene transcript compared to all otherconditions was not noted in the condition in which BMP-6 was added tothe culture along with TGF-β3 and IGF-I.

The addition of BMP-6 and TGF-β3 alone also resulted in a significantincrease in col1a1 gene expression over Day 0 controls (p<0.05).Compared to the control medium control group at seven days, the cocktailincluding TGF-β3, IGF-I, and BMP-6 resulted in significant upregulationof collal gene expression (p<0.0001), whereas the addition of BMP-6alone did not. All conditions containing TGF-β1 or TGF-β3 and not BMP-6demonstrated decreased COL1A1 gene expression when compared to thecocktail containing TGF-β3, IGF-I, and BMP-6 (p<0.0001). Interestingly,however, no significant differences were observed in comparing thesesame TGF-β conditions to the BMP-6 alone condition. Along these samelines, the cocktails containing TGF-β3 and IGF-I as well as the groupcontaining TGF-β3, IGF-I, and BMP-6 were statistically different thanthe condition containing BMP-6 alone (p<0.05).

Compared to Day 0 controls, the addition of BMP-6 in the two conditionsor the condition containing TGF-β3 alone resulted in a significantupregulation of col2a1 gene expression (p<0.05). However, of these threeconditions, only the condition containing TGF-β3, IGF-I, and BMP-6resulted in a significant increase in gene expression relative to theDay 7 control medium condition (p=0.0017). The two BMP-6 conditions alsoresulted in a significant upregulation of COL2A1 compared to either ofthe conditions containing TGF-β1 (p<0.001). Comparing TGF-β1 and TGF-β3,TGF-β3 shows a significant increase in COL2A1 gene expression relativeto TGF-β1+dexamethasone and relative to TGF-β3+IGF-I suggesting thateither dexamethasone or IGF-I inhibits COL2A1 when used in combinationwith TGF-β3 (p<0.05). Interestingly, the addition of BMP-6 restored theinhibitory nature of IGF-I (p<0.05).

For COL10A1 gene expression, all of the conditions containing a TGF-βisoform resulted in a significant increase in COL10A1 mRNA transcriptlevels compared to either the Day 0 control cells or the control mediumat Day 7. Conversely, the addition of exogenous BMP-6 significantlyreduced the levels of COLA10A1 transcript levels after seven days inculture relative to the Day 0 control cells and the TGF-β isoformconditions.

Example 4 Immunohistochemistry

The same trends existing in the gene expression data were also evidentin the immunohistochemistry results. The negative controls exhibitedminimal background staining. In addition, the immunohistochemistryresults from the control showed insignificant staining for allantibodies studied. The most robust and interesting trends were seen inthe TGF-β1+dexamethasone and the two BMP-6 groups as shown in FIG. 7.

Specifically, more intense staining for chondroitin 6-sulfate (3B3epitope) is seen with the BMP-6 group compared to theTGF-β1+dexamethasone condition. This same trend was also observed withthe collagen II antibody (II-II6B3). The expression of collagen I showedlittle qualitative differences across the groups, though collagen I isnoted in the pericellular and extracellular matrix with all the growthfactors employed; whereas the addition of BMP-6 alone resulted in asignificant decrease in staining intensity for collagen X compared tothe TGF-β1+dexamethasone condition.

Discussion of Examples 1-4

The effects of BMP-6 on hADS cells have not previously been described.The presently disclosed subject matter shows that BMP-6 is a stronginducer of a phenotype that has some cartilage characteristics in hADScells compared to other growth factors. For aggrecan gene expression,the addition of BMP-6 alone, averaged across the three cell donors,resulted in a 205-fold increase in aggrecan gene expression (FIG. 3).Somewhat surprisingly, the control group, which included the base mediumwith only 10% FBS, showed consistent increases in aggrecan geneexpression roughly equal (on average) to the other growth factors usedin this study. Statistically significant differences were noted withCOL1A1 gene expression between the conditions containing BMP-6 and theother conditions.

Interestingly, the condition containing TGF-β3, IGF-I, and BMP-6resulted in a significant increase in COL1A1 gene expression relative toother conditions, while BMP-6 alone, compared to BMP-6 in conjunctionwith the other two growth factors, showed a decrease in COL1A1 geneexpression potentially alluding to synergism between TGF-β3, IGF-I, andBMP-6 in promoting col1a1 gene expression (FIG. 4). BMP-6 alsoconsistently increased COL2A1 gene expression across all 3 donors; thiswas seen in both the multiple growth factor condition containing BMP-6as well as BMP-6 alone. The other growth factor combinations were ableto induce COL2A1 gene expression in two of the three donors. Again, andsomewhat surprisingly, the base medium was also able to induce aconsistent increase in COL2A1 gene expression, though this should beviewed in light of a significant decrease in DNA content for this basemedium condition over the seven-day time course (FIG. 1).

In stark contrast to previous studies in the art (Sekiva et al. (2001)Biochem Biophys Res Commun 284:411-418; Sekiva et al. (2002) Proc NatlAcad Sci USA 99:4397-4402; Indrawattana et al. (2004) Biochem BiophysRes Commun 320:914-919), the results presented herein not onlydemonstrate BMP-6 as a strong inducer of two chondrogenic markers,aggrecan and COL2A1, but also a strong inhibiter of thehypertrophic/endochondral ossification pathway as measured bysignificant decreases in COL10A1 gene expression and COL10A1 antibodystaining compared to other conditions (FIG. 6 and FIG. 7). One mightargue that longer time periods in this study would reveal an increase inCOL10A1 gene expression; however, it should be noted that even at sevendays in the studies by Sekiya et al., (Sekiva et al. (2001) BiochemBiophys Res Commun 284:411-418; Sekiva et al. (2002) Proc Natl Acad SciUSA 99:4397-4402 Sekiya et al., 2001; Sekiya et al., 2002), PCR analysisrevealed an increase in COL10A1 gene expression relative to the Day 0controls, which is directly opposite the results disclosed herein.Again, it should be noted that this decrease in the COL10A1 geneexpression was also observed in the immunohistochemistry data (e.g.,compare FIGS. 7F to 7D and 7E).

Exchanging TGF-β1 for TGF-β3 also did not seem to have a profound effectin promoting a differentiation effect as both isotypes exhibit similarresponses across all assays; this result is somewhat inconsistent withother work, which showed that TGF-β3 was superior to TGF-β1 in inducingchondrogenesis in MSCs (Barry et al. (2001) Exp Cell Res 268:189-200).IGF-I also did not seem to have a strong effect in promoting synergismwith TGF-β3 and with TGF-β3 and BMP-6; in fact the addition of IGF-I andTGF-β3 to BMP-6 appears to partially inhibit the response of the cellsto BMP-6. One potential explanation would be that both TGF-β3 and IGF-Ican initiate multiple signaling pathways different from that of BMP-6and that downstream events associated with these pathways can somehowcompete and inhibit the ability of BMP-6 to promote differentiation.

The most widely used growth cocktail for inducing chondrogenesisincludes TGF-β1 and dexamethasone (Johnstone et al. (1998) Exp Cell Res238:265-272) as discussed herein. While this combination of growthfactors is able to induce a chondrogenic response in other mesenchymalstem cells, the presently disclosed subject matter demonstrates thatthis combination of growth factors is less than ideal for promotingchondrogenesis in hADS cells. This condition showed the highest DNAcontent at Day 7 relative to Day 0 with the levels of DNA stayingconsistent on average with Day 0 DNA levels (FIG. 1). ThisTGF-β1+dexamethasone condition also showed the highest [³H]-prolinebiosynthesis rates over the last 24 hours of culture, indicatingmetabolically active cells.

Although the highest rates of protein synthesis and highest levels ofDNA content are observed in the TGF-β1+dexamethasone condition, thiscondition proved to be the weakest inducer of COL2A1 mRNA expression,which was also observed in the collagen 2 immunohistological analysis.Increases in both COL1A1 and COL10A1 gene expression along with aconcomitant decrease in both aggrecan gene expression and 3B3immunohistological staining suggest that this combination can induce anosteogenic phenotype.

Comparatively, the BMP-6 conditions also maintained higher DNA amountsand biosynthetic activities after seven days across the three donors ascompared to the other conditions without dexamethasone (FIGS. 1 and 2).A decrease in COL2A1 gene expression when dexamethasone is added toTGF-β1, compared to the TGF-β1 condition alone, suggests thatdexamethasone is somewhat inhibiting COL2A1.

Studies by Boden et al. (Boden et al. (1996) Endocrinology137:3401-3407; Boden et al. (1997) Endocrinology 138:2820-2828; Boden etal. (1998) Endocrinology 139:5125-5134; Liu et al. (2004) Bone35:673-681) indicate that BMP-6 is also upregulated by glucocorticoids,suggesting that BMP-6 is required for endochondral ossification andplays a role in bone formation in MSCs. Conversely, the data disclosedherein suggest that BMP-6 does not signal through the same pathway as inMSCs since a different response is observed than that reported for MSCs.

As disclosed herein, hADS cells, respond in a vastly different fashionthan MSCs. Most notably and in direct contrast to the effects BMP-6 hason other cell types in inducing a strong osteogenic phenotype, not onlydoes BMP-6 induce a novel phenotype as indicated by strong COL2A1 andaggrecan gene expression and immunohistochemical data, it also appearsto inhibit the endochondral ossification pathway as evidenced by thesignificant decrease in COL10A1 gene expression, also confirmed byimmunohistochemistry.

As new paradigms for clinical intervention for musculoskeletal tissuepathology are sought, the present disclosure suggests that hADS cellsused in conjunction with BMP-6 can be viable candidates for variousremodeling, repair, regrowth, and/or regeneration strategies of variousorthopaedic tissues, which serve mechanical functions. Some embodimentsinclude the use of hADS cells that have been induced ex vivo to producecartilaginous-like tissue and then reimplanted. Some embodiments includea genetic engineering approach in which the gene for either BMP-6 or theBMP-6 receptor could be inserted into the genome of hADS cells and thendelivered to the pathological site for in vivo repair/regeneration.

References

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thepresently disclosed subject matter. Furthermore, the foregoingdescription is for the purpose of illustration only, and not for thepurpose of limitation.

What is claimed is:
 1. A method of resurfacing a predetermined articularjoint in a subject, the joint having a number of focal defects, themethod comprising: (a) providing an implant comprising: (i) a scaffoldcomprising a woven fibrous biocompatible material sized to replacesubstantially all of the articulating surface of the predeterminedjoint, and shaped substantially like said articulating surface of saidpredetermined joint; and (ii) one or more adipocyte stem cells ; (b)infiltrating the one or more of said cells throughout the scaffold; and(c) implanting at a site of the joint in the subject the implantprovided in steps (a) and (b) to thereby resurface said articulatingsurface of the joint in the subject encompassing the number of focaldefects.
 2. A method of resurfacing a predetermined articular joint in asubject, the joint having a number of focal defects, the methodcomprising: (a) providing an implant comprising: (i) a scaffoldcomprising a woven fibrous biocompatible material and sized to replacesubstantially all of the articulating surface of the predetermined jointand shaped substantially like said articulating surface of saidpredetermined joint; and (ii) one or more adipocyte stem cells; (b)encouraging infiltration of the one or more cells throughout thescaffold; (c) devitalizing a fraction of the cells before implantation;and (d) implanting at a site of the joint in the subject the implantprovided in steps (a), (b), and (c) to thereby resurface saidarticulating surface of the joint in the subject encompassing the numberof focal defects.
 3. The method of any one of claims 1, and 2, whereinthe biocompatible material comprises a material selected from the groupconsisting of an absorbable material, a non-absorbable material, andcombinations thereof.
 4. The method of claim 3, wherein thenon-absorbable material is selected from the group consisting of apolytetrafluoroethylene (PTFE), an expanded PTFE (ePTFE), a polyamide, anylon, a polysulfone, a cellulosic, an acrylic, tantalum, polyvinylalcohol, carbon, ceramic, a metal, an acrylic, a polycarbonate, apolyester, a polyether, a poly(ether ketone), a poly(ether etherketone), a poly(aryl ether ketone), a poly(ether ether ketone etherketone), a poly(ethylene terephthalate), a poly(methyl (meth)acrylate),a polyolefin, a polysulfone, a polyurethane, a polyethylene, apolypropylene, a poly(vinyl chloride), a carbon fiber reinforcedcomposite, a glass fiber reinforced composite, and combinations thereof.5. The method of claim 3, wherein the absorbable material is selectedfrom the group consisting of a polyglycolic acid (PGA), a polylacticacid (PLA), a polyglycolide-Iactide, a polycaprolactone, apolydioxanone, a polyoxalate, a polyanhydride, a poly(phosphoester),catgut suture, collagen, silk, agarose, chitin, chitosan,hydroxyapatite, bioabsorbable calcium phosphate, hyaluronic acid,elastin, a polyorthoester, a poly(amino acid), a pluronic/F-12, apoly(ethylene oxide)/poly(ethylene glycol) (PEG/PEG), collagen, fibrin,hyaluronic acid, a proteoglycan, elastin, and combinations thereof. 6.The method of any one of claims 1, and 2, wherein the scaffold comprisesthree-dimensionally woven biocompatible fibers.
 7. The method of claim6, wherein the fibers comprise a monofilament fiber, a multifilamentfiber, a hollow fiber, a fiber having a variable cross-section along itslength, or a combination thereof.
 8. The method of claim 6, wherein athree-dimensional fiber scaffold is utilized, comprising threeorthogonally woven fiber systems, a plurality of braided fiber systems,a plurality of circular woven fiber systems, or combinations thereof. 9.The method of claim 6, wherein the scaffold comprises athree-dimensional fiber scaffold, the scaffold comprising at least threesystems of fibers, wherein (i) two of the three fiber systems define anupper layer, a lower layer, and a medial layer between the upper layerand the lower layer within the three-dimensional fiber scaffold; (ii)one of the at least three fiber systems interconnects the upper layer,the lower layer and the medial layer; and (iii) the at least three fibersystems each comprise a biocompatible material.
 10. The method of claim9, wherein the at least three fiber systems in at least one of theupper, medial, and lower layers define a plurality of interstices withinthe fiber scaffold.
 11. The method of claim 10, wherein the intersticescomprise a pore size ranging from about 1 μm to about 1,000 μm ,optionally from about 10 μm to about 500 μm, optionally from about 25 μmto about 250 μm, or optionally, from about 50 μm to about 125 μm. 12.The method of any one of claims 1, and 2, comprising providing theimplant in a shape that corresponds to a majority of said articulatingsurface of the predetermined joint.
 13. The method of claim 12, whereinthe shape is substantially that of the native predetermined joint. 14.The method of any one of claims 1, and 2, wherein one or more surfacesof the scaffold are coated with a biomaterial layer.
 15. The method ofclaim 14, wherein the biomaterial layer comprises a gel.
 16. The methodof any one of claims 1, and 2, further comprising providing abiologically active material to the implant.
 17. The method of claim 16,wherein the biologically active material is selected from the groupconsisting of a growth factor, a cytokine, a chemokine, a collagen,gelatin, laminin, fibronectin, thrombin, lipids, cartilage oligomericprotein (COMP), thrombospondin, fibrin, fibrinogen, Matrix-GLA(glycine-leucine-alanine) protein, chondrocalcin, tenascin, a mineral,an RGD (Arginine-Glycine-Aspartic Acid) peptide or RGD-peptidecontaining molecule, elastin, hyaluronic acid, a glycosaminoglycan, aproteoglycan, water, an electrolyte solution, and combinations thereof.18. The method of any one of claims 1, and 2, wherein the predeterminedjoint is selected from the group consisting of a hip joint, a kneejoint, a shoulder joint, an ankle joint, thumb joint, finger joint,wrist joint, neck joint, spine joint, toe joint, temporomandibularjoint, patella, and an elbow joint.
 19. The method of any one of claims1 and 2, comprising maintaining the joint resurfacing implant in abioreactor prior to implantation for a time sufficient to provide tissuethat can resurface at least a portion of an articulating surface of thepredetermined joint.
 20. The method of any one of claims 1, and 2,comprising removing part or all tissues present at site of the joint.21. The method of claim 20, comprising removing tissues selected fromthe group consisting of cartilage, bone, ligaments, meniscus, synovium,and combinations thereof.
 22. The method of any one of claims 1, and 2,comprising resurfacing an entire articulating surface of the joint. 23.The method of any one of claims 1, and 2, wherein at least a portion oftwo or more articulating surfaces of the joint are resurfaced in part orin all.
 24. The method of claim 23, wherein at least a portion of allarticulating surfaces of the joint are resurfaced.
 25. The method ofclaim 24, comprising completely resurfacing all articulating surfaces ofthe joint.